Sugarcane UBI9 gene promoter and methods of use thereof

ABSTRACT

The invention relates to the nucleotide sequence from sugarcane ubi9 polyubiquitin gene promoter, which is capable of directing constitutive expression of a nucleic acid sequence of interest that is operably linked to it. The sugarcane ubi9 promoter is useful in regulating expression of a nucleic acid sequence of interest in monocotyledonous and dicotyledonous plants.

“This application claims priority to now abandoned provisionalapplication: 60/078,768, filed on Mar. 19, 1998.”

This work was made with Government support by the United StatesDepartment of Agriculture. The Government has certain rights in thisinvention.

FIELD OF THE INVENTION

The invention relates to nucleic acid sequences isolated from sugarcaneand to methods of using them. In particular, the inventions relates tonucleotide sequences which are derived from sugarcane polyubiquitingenes and which are capable of directing constitutive expression of anucleic acid sequence of interest that is operably linked to thesugarcane polyubiquitin nucleotide sequences. The sugarcanepolyubiquitin nucleotide sequences are useful in regulating expressionof a nucleic acid sequence of interest in monocotyledonous anddicotyledonous plants.

BACKGROUND OF THE INVENTION

Much scientific effort has been directed at genetically engineeringplants to produce agronomically relevant proteins. Recombinant genes forproducing proteins in plants require a promoter sequence which iscapable of directing protein expression in plant cells. Promotersequences which direct high levels of protein expression in plant cellsare particularly desirable since fewer numbers of transgenic plants needto be produced and screened to recover plants producing agronomicallysignificant quantities of the target protein. In addition, high levelsof protein expression aid the generation of plants which exhibitcommercially important phenotypic properties, such as pest and diseaseresistance, resistance to environmental stress (e.g., water-logging,drought, heat, cold, light-intensity, day-length, chemicals, etc.),improved qualities (e.g., high yield of fruit, extended shelf-life,uniform fruit shape and color, higher sugar content, higher vitamins Cand A content, lower acidity, etc.).

Some promoter sequences which are capable of driving expression oftransgenes (e.g., selectable marker genes) in plants are known in theart and are derived from a variety of sources such as bacteria, plantDNA viruses, and plants. Promoters of bacterial origin include theoctopine synthase promoter, the nopaline synthase promoter and otherpromoters derived from native Ti plasmids. Promoters of viral origininclude the 35S and 19S RNA promoters of cauliflower mosaic virus. Plantpromoters include the ribulose-1,3-diphosphate carboxylase small subunitpromoter, the phaseolin promoter, and the maize polyubiquitin promoter.

While some promoter sequences which function in plant cells areavailable, expression of more than one gene (e.g., a selectable markergene and an agronomically relevant gene) which is operably linked to thesame promoter sequence is likely to be hampered by homology dependentsilencing of transgenes in plants. Thus, what is needed are additionalpromoter sequences which are capable of driving transgene expression. Inparticular, what is needed are promoter sequences which drive transgeneexpression in both monocotyledonous and dicotyledonous plant cells.

SUMMARY OF THE INVENTION

The present invention provides nucleic acid sequences having promoteractivity. The nucleic acid sequences provided herein direct expressionof operably linked nucleotide sequences in cells, tissues and organs ofmonocotyledonous and dicotyledonous plants. In one embodiment, theinvention provides a substantially purified nucleic acid sequencecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:7, the complement of SEQ ID NO:7, homologs of SEQ ID NO:7,homologs of the complement of SEQ ID NO:7; SEQ ID NO:10, the complementof SEQ ID NO:10, homologs of SEQ ID NO:10, and homologs of thecomplement of SEQ ID NO:10. In a preferred embodiment, the nucleotidesequence is characterized by having promoter activity. In a morepreferred embodiment, the promoter activity is constitutive. In anotherembodiment, nucleotide sequence is double-stranded. In yet anotherembodiment, the nucleotide sequence is single-stranded. In yet anotheralternative embodiment, the nucleic acid sequence is contained in aplant cell. In a preferred embodiment, the plant cell is derived from amonocotyledonous plant. In a more preferred embodiment, themonocotyledonous plant is selected from the group consisting ofsugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye,yam, onion, banana, coconut, date, and hop. In an alternative morepreferred embodiment, the plant cell is derived from a dicotyledonousplant. In a preferred embodiment, the dicotyledonous plant is selectedfrom the group consisting of tobacco, tomato, soybean, and papaya.

The invention also provides a substantially purified nucleic acidsequence comprising a portion of a nucleotide sequence selected from thegroup consisting of SEQ ID NO:7 and the complement thereof. In apreferred embodiment, the portion is characterized by having promoteractivity. In a more preferred embodiment, the promoter activity isconstitutive. In an alternative preferred embodiment, the portion isdouble-stranded. In another alternative preferred embodiment, theportion is single-stranded. In yet another alternative preferredembodiment, the portion comprises the nucleotide sequence selected fromthe group consisting of the nucleotides from 1 to 242, from 245 to 787,from 788 to 1020, from 1021 to 1084, from 1085 to 1168, from 1169 to1173, from 1174 to 1648, from 1649 to 1802, from 1 to 377, from 378 to442, and from 443 to 1802. In a further alternative preferredembodiment, nucleic acid sequence is contained in a plant cell. In amore preferred embodiment, the plant cell is derived from amonocotyledonous plant. In a yet more preferred embodiment, themonocotyledonous plant is selected from the group consisting ofsugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye,yam, onion, banana, coconut, date, and hop. In another more preferredembodiment, the plant cell is derived from a dicotyledonous plant. In ayet more preferred embodiment, the dicotyledonous plant is selected fromthe group consisting of tobacco, tomato, soybean, and papaya.

Further provided by the invention is a substantially purified nucleicacid sequence comprising a portion of a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:10 and the complement thereof. Ina preferred embodiment, the portion is characterized by having promoteractivity. In a more preferred embodiment, the promoter activity isconstitutive. In an alternative preferred embodiment, the portion isdouble-stranded. In another alternative preferred embodiment, theportion is single-stranded. In yet another alternative preferredembodiment, the portion comprises the nucleotide sequence selected fromthe group consisting of the nucleotides 1 to 3600, from 3602 to 3612,from 3614 to 3688, from 1 to 2248, from 2249 to 2313, from 2314 to 3688,and from 1671 to 2248. In another alternative preferred embodiment, thenucleic acid sequence is contained in a plant cell. In a more preferredembodiment, the plant cell is derived from a monocotyledonous plant. Inyet a more preferred embodiment, the monocotyledonous plant is selectedfrom the group consisting of sugarcane, maize, sorghum, pineapple, rice,barley, oat, wheat, rye, yam, onion, banana, coconut, date, and hop. Inan alternative more preferred embodiment, the plant cell is derived froma dicotyledonous plant. In a yet more preferred embodiment, thedicotyledonous plant is selected from the group consisting of tobacco,tomato, soybean, and papaya.

The invention additionally provides a substantially purified nucleicacid sequence comprising the EcoRI/XbaI fragment isolated from plasmidpubi4-GUS contained in Escherichia coli cells deposited as NRRLB-30115,the complement of the fragment, homologs of the fragment, and homologsof the complement of the fragment. In a preferred embodiment, thenucleotide sequence is SEQ ID NO:7. In a more preferred embodiment, thenucleotide sequence is characterized by having promoter activity. In ayet more preferred embodiment, the promoter activity is constitutive. Inan alternative yet more preferred embodiment, the nucleotide sequence isdouble-stranded. In another alternative more preferred embodiment, thenucleotide sequence is single-stranded. In yet another alternative morepreferred embodiment, the nucleic acid sequence is contained in a plantcell. In another preferred embodiment, the plant cell is derived from amonocotyledonous plant. In a more preferred embodiment, themonocotyledonous plant is selected from the group consisting ofsugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye,yam, onion, banana, coconut, date, and hop. In an alternative preferredembodiment, the plant cell is derived from a dicotyledonous plant. In ayet more preferred embodiment, the dicotyledonous plant is selected fromthe group consisting of tobacco, tomato, soybean, and papaya.

Also provided herein is a substantially purified nucleic acid sequencecomprising the HindIII/XbaI fragment isolated from plasmid pubi9-GUScontained in Escherichia coli cells deposited as NRRLB-30116, thecomplement of the fragment, homologs of the fragment, and homologs ofthe complement of the fragment. In a preferred embodiment, thenucleotide sequence is SEQ ID NO:10. In an alternative preferredembodiment, the nucleotide sequence is characterized by having promoteractivity. In a more preferred embodiment, the promoter activity isconstitutive. In another alternative preferred embodiment, thenucleotide sequence is double-stranded. In yet another alternativepreferred embodiment the nucleotide sequence is single-stranded. Inanother alternative preferred embodiment, the nucleic acid sequence iscontained in a plant cell.In a more preferred embodiment, the plant cellis derived from a monocotyledonous plant. In a yet more preferredembodiment, the monocotyledonous plant is selected from the groupconsisting of sugarcane, maize, sorghum, pineapple, rice, barley, oat,wheat, rye, yam, onion, banana, coconut, date, and hop. In another morepreferred embodiment, the plant cell is derived from a dicotyledonousplant. In a yet more preferred embodiment, the dicotyledonous plant isselected from the group consisting of tobacco, tomato, soybean, andpapaya.

The invention further provides a substantially purified nucleic acidsequence comprising a portion of the EcoRI/XbaI fragment isolated fromplasmid pubi4-GUS contained in Escherichia coli cells deposited asNRRLB-30115, and the complement of the fragment. In a preferredembodiment, the portion is characterized by having promoter activity. Ina more preferred embodiment, the promoter activity is constitutive. Inan alternative preferred embodiment, the portion is double-stranded. Inanother alterative preferred embodiment, the portion is single-stranded.In yet another alternative preferred embodiment, the nucleic acidsequence is contained in a plant cell. In a more preferred embodiment,the plant cell is derived from a monocotyledonous plant.In a yet morepreferred embodiment, the monocotyledonous plant is selected from thegroup consisting of sugarcane, maize, sorghum, pineapple, rice, barley,oat, wheat, rye, yam, onion, banana, coconut, date, and hop. In analternative more preferred embodiment, the plant cell is derived from adicotyledonous plant. In yet a more preferred embodiment, thedicotyledonous plant is selected from the group consisting of tobacco,tomato, soybean, and papaya.

Also provided by the invention is a substantially purified nucleic acidsequence comprising a portion of the HindIII/XbaI fragment isolated fromplasmid pubi9-GUS contained in Escherichia coli cells deposited asNRRLB-30116, and the complement of the fragment. In a preferredembodiment, the portion is characterized by having promoter activity. Ina more preferred embodiment, the promoter activity is constitutive. Inanother preferred embodiment, the portion is double-stranded. In yetanother preferred embodiment, the portion is single-stranded. In yetanother preferred embodiment, the nucleic acid sequence is contained ina plant cell. In a more preferred embodiment, the plant cell is derivedfrom a monocotyledonous plant. In a yet more preferred embodiment, themonocotyledonous plant is selected from the group consisting ofsugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye,yam, onion, banana, coconut, date, and hop. In an alterative morepreferred embodiment, the plant cell is derived from a dicotyledonousplant. In a yet more preferred embodiment, the dicotyledonous plant isselected from the group consisting of tobacco, tomato, soybean, andpapaya.

The invention additionally provides a recombinant expression vectorcomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:7, the complement of SEQ ID NO:7, homologs of SEQ ID NO:7,homologs of the complement of SEQ ID NO:7; SEQ ID NO:10, the complementof SEQ ID NO:10, homologs of SEQ ID NO:10, and homologs of thecomplement of SEQ ID NO:10. In a preferred embodiment, the recombinantexpression vector is selected from the group consisting of pubi4-GUS,pubi9-GUS, 4PI-GUS and 9PI-GUS.

The invention also provides a recombinant expression vector comprising aportion of a nucleotide sequence selected from the group consisting ofSEQ ID NO:7 and the complement thereof.

Also provided herein is a recombinant expression vector comprising aportion of a nucleotide sequence selected from the group consisting ofSEQ ID NO:10 and the complement thereof.

Additionally provided by the invention is a transgenic plant cellcomprising a nucleic acid sequence comprising a nucleotide sequenceselected from the group consisting of SEQ ID NO:7, the complement of SEQID NO:7, homologs of SEQ ID NO:7, homologs of the complement of SEQ IDNO:7; SEQ ID NO:10, the complement of SEQ ID NO:10, homologs of SEQ IDNO:10, and homologs of the complement of SEQ ID NO:10, wherein thenucleotide sequence is operably linked to a nucleic acid sequence ofinterest. In a preferred embodiment, the transgenic plant cell expressesthe nucleic acid sequence of interest. In a more preferred embodiment,the expression is constitutive. In an alternative embodiment, thetransgenic plant cell is derived from a monocotyledonous plant. In amore preferred embodiment, the monocotyledonous plant is selected fromthe group consisting of sugarcane, maize, sorghum, pineapple, rice,barley, oat, wheat, rye, yam, onion, banana, coconut, date, and hop. Inanother alternative embodiment, the transgenic plant cell is derivedfrom a dicotyledonous plant. In a more preferred embodiment, thedicotyledonous plant is selected from the group consisting of tobacco,tomato, soybean, and papaya. In yet another alternative embodiment, thenucleic acid sequence of interest is a sense sequence. In a morepreferred embodiment, the sense sequence encodes a protein selected fromthe group consisting of β-glucuronidase, luciferase, β-galactosidase,1-aminocyclopropane-1-carboxylic acid deaminase, sucrose phosphatesynthase, 5-enolpyruvyl-3- phosphoshikimate synthase, acetolactatesynthase, RNase, wheat germ agglutinin, sweetness protein, and Bacillusthuringiensis crystal toxin proteins. In a further alternativeembodiment, the nucleic acid sequence of interest is an antisensesequence. In a more preferred embodiment, the antisense sequence isselected from the group consisting of an antisense sequence to ACCsynthase, to ethylene inducible sequences, and to polyphenol oxidase.

The invention further provides a transgenic plant cell comprising anucleic acid sequence comprising a portion of a nucleotide sequenceselected from the group consisting of SEQ ID NO:7 and the complementthereof.

Also provided herein is a transgenic plant cell comprising a nucleicacid sequence comprising a portion of a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:10 and the complement thereof.

Additionally provided by the invention is a method for expressing anucleic acid sequence of interest in a plant cell, comprising: a)providing: i) a plant cell; ii) a nucleic acid sequence of interest; andiii) a nucleotide sequence selected from the group consisting of SEQ IDNO:7, the complement of SEQ ID NO:7, homologs of SEQ ID NO:7, homologsof the complement of SEQ ID NO:7; SEQ ID NO:10, the complement of SEQ IDNO:10, homologs of SEQ ID NO:10, and homologs of the complement of SEQID NO:10; b) operably linking the nucleic acid sequence of interest tothe nucleotide sequence to produce a transgene; and c) introducing thetransgene into the plant cell to produce a transgenic plant cell underconditions such that the nucleic acid sequence of interest is expressedin the transgenic plant cell. In a preferred embodiment, the methodfurther comprises d) identifying the transgenic plant cell. In anotherpreferred embodiment, the method further comprises d) regeneratingtransgenic plant tissue from the transgenic plant cell. In analternative preferred embodiment, the methods further comprises d)regenerating a transgenic plant from the transgenic plant cell. Inanother preferred embodiment, the plant cell is derived from amonocotyledonous plant. In yet a more preferred embodiment, themonocotyledonous plant is selected from the group consisting ofsugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye,yam, onion, banana, coconut, date, and hop. In another alterative morepreferred embodiment the plant cell is derived from a dicotyledonousplant. In a yet more preferred embodiment, the dicotyledonous plant isselected from the group consisting of tobacco, tomato, soybean, andpapaya.

The invention further provides a method for expressing a nucleic acidsequence of interest in a plant cell, comprising: a) providing: i) aplant cell; ii) a nucleic acid sequence of interest; and iii) a portionof a nucleotide sequence selected from the group consisting of SEQ IDNO:7 and the complement thereof; b) operably linking the nucleic acidsequence of interest to the portion of a nucleotide sequence to producea transgene; and c) introducing the transgene into the plant cell toproduce a transgenic plant cell under conditions such that the nucleicacid sequence of interest is expressed in the transgenic plant cell. Ina preferred embodiment, the portion comprises the nucleotide sequenceselected from the group consisting of the nucleotides from 1 to 242,from 245 to 787, from 788 to 1020, from 1021 to 1084, from 1085 to 1168,from 1169 to 1173, from 1174 to 1648, from 1649 to 1802, from 12 to 377,from 378 to 442, and from 443 to 1802.

Also provided by the invention is a method for expressing a nucleic acidsequence of interest in a plant cell, comprising: a) providing: i) aplant cell; ii) a nucleic acid sequence of interest; and iii) a portionof a nucleotide sequence selected from the group consisting of SEQ IDNO:10 and the complement thereof; b) operably linking the nucleic acidsequence of interest to the portion of a nucleotide sequence to producea transgene; and c) introducing the transgene into the plant cell toproduce a transgenic plant cell under conditions such that the nucleicacid sequence of interest is expressed in the transgenic plant cell. Ina preferred embodiment, the portion comprises the nucleotide sequenceselected from the group consisting of the nucleotides 1 to 3600, from3602 to 3612, from 3614 to 3688, from 1 to 2248, from 2249 to 2313, from2314 to 3688, and from 1671 to 2248.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a RNA gel blot of sugarcane polyubiquitin mRNA poolshybridized to gene-specific probes for Scubi 221, Scubi 511, Scubi 241,Scubi561, and Scubi 5121.

FIG. 2 shows a “Reverse northern” blot of sugarcane polyubiquitin mRNApools.

FIGS. 3(A-B) shows the nucleotide sequence of the sugarcanepolyubiquitin ubi4 gene. FIG. 3A shows the nucleotide sequence (SEQ IDNO:1) including the translation start codon and sequences upstreamthereof. FIG. 3B shows the nucleotide sequence (SEQ ID NO:2) includingthe translation stop codon and sequences downstream thereof. Boldfaceindicates putative TATA box, ATG translation initiation codon, TAAtranslation stop codon, and putative poly-A addition signal; underliningindicates the putative 5′ and 3′ UTRs, based on homology to scubi241 and511 cDNAs; lowercase indicates that the base is as depicted but withsome uncertainty (see also Tables 1 and 2 below).

FIG. 4 is a diagrammatic representation of the initially-determinedorganization of ubi4 and ubi9 polyubiquitin genes. Numbered white boxesindicate polyubiquitin coding repeats, unnumbered white boxes are 5′ and3′ untranslated regions, black boxes are introns, and lines are flankingDNA. E: EcoRI; N: NruI; S: SalI; H: HindIII; M: putative miniatureinverted-repeat transposable element.

FIGS. 5(A-B) shows the nucleotide sequence (SEQ ID NO:5) (nucleotides 1to 5512 of the 5551 nucleotide sequence deposited as GenBank accessionnumber AF093504) (A) and translated amino acid sequence (SEQ ID NO:6)(B) of sugarcane polyubiquitin ubi4 gene.

FIG. 6 is a diagrammatic representation of the organization of ubi4(GenBank accession number AF093504) and ubi9 (GenBank accession numberAF093505) polyubiquitin genes. Numbered white boxes indicatepolyubiquitin coding repeats, unnumbered white boxes are 5′ and 3′untranslated regions, black boxes are introns and lines are flankingDNA. E: EcoRI; N: NruI; S: SalI; H: HindIII; M: putative miniatureinverted-repeat transposable element.

FIGS. 7(A-B) shows the nucleotide sequence of the sugarcanepolyubiquitin ubi9 gene. FIG. 7A shows the nucleotide sequence (SEQ IDNO:3) including the translation start codon and sequences upstreamthereof. FIG. 7B shows the nucleotide sequence (SEQ ID NO:4) includingthe translation stop codon and sequences downstream thereof. Boldfaceindicates the putative TATA box, ATG translation start codon, TAAtranslation stop codon, and putative poly-A addition signal; underliningindicates the 5′ and 3′ UTRs; lowercase indicates that the base is mostprobably as depicted (see also Tables 4 and 5 below).

FIGS. 8(A-B) shows the nucleotide sequence (SEQ ID NO:8) (GenBankaccession number AF093505) (A) and translated amino acid sequence (SEQID NO:9) (B) of the sugarcane polyubiquitin ubi9 gene.

FIGS. 9(A-D) is a graph showing GUS activity following transientexpression of GUS under the control of the sugarcane ubi4 promoter(ubi4), sugarcane ubi9 promoter (ubi9), and maize ubi1 promoter (mzubi1)in sugarcane suspension cells (A, B) and tobacco leaves (C, D). Controlsreceived no DNA. Letters within parentheses indicate least significantdifference levels, i.e., mean GUS activity values with different lettersbeing significantly different at P≦0.05 confidence level.

FIG. 10 shows the nucleotide sequence (SEQ ID NO:7) of the portion ofthe ubi4 gene which was ligated to the gene encoding β-glucuronidase(GUS) in plasmids pubi4-GUS and 4PI-GUS. SEQ ID NO:7 corresponds tonucleotides 1-1802 of SEQ ID NO:5.

FIG. 11 shows the nucleotide sequence (SEQ ID NO:10) of the portion ofthe ubi9 gene which was ligated to the gene encoding β-glucuronidase(GUS) in plasmids pubi9-GUS and 9PI-GUS. SEQ ID NO:10 corresponds tonucleotides 1-3688 of SEQ ID NO:8.

FIGS. 12(A-D) is a graph showing GUS activity following stableexpression in sugarcane callus lines of GUS under the control ofsugarcane ubi9 promoter (sc ubi9) (A, D), sugarcane ubi4 promoter (scubi4) (B, D), and maize ubi1 (C, D).

FIG. 13 is a graph showing GUS reporter gene activity in stabletransgenic rice callus lines expressed under the control of thesugarcane ubi9 promoter in the presence and absence of the putativenuclear matrix attachment region (MAR).

FIG. 14 shows the nucleotide sequence (SEQ ID NO:11) encoding polyphenoloxidase (GenBank accession number s40548).

FIG. 15 shows the nucleotide sequence (SEQ ID NO:2) encoding maizesucrose phosphate synthase enzyme (GenBank accession number m97550).

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

The term “transgenic” when used in reference to a cell refers to a cellwhich contains a transgene, or whose genome has been altered by theintroduction of a transgene. The term “transgenic” when used inreference to a tissue or to a plant refers to a tissue or plant,respectively, which comprises one or more cells that contain atransgene, or whose genome has been altered by the introduction of atransgene. Transgenic cells, tissues and plants may be produced byseveral methods including the introduction of a “transgene” comprisingnucleic acid (usually DNA) into a target cell or integration of thetransgene into a chromosome of a target cell by way of humanintervention, such as by the methods described herein.

The term “transgene” as used herein refers to any nucleic acid sequencewhich is introduced into the genome of a cell by experimentalmanipulations. A transgene may be an “endogenous DNA sequence,” or a“heterologous DNA sequence” (i.e., “foreign DNA”). The term “endogenousDNA sequence” refers to a nucleotide sequence which is naturally foundin the cell into which it is introduced so long as it does not containsome modification (e.g., a point mutation, the presence of a selectablemarker gene, etc.) relative to the naturally-occurring sequence. Theterm “heterologous DNA sequence” refers to a nucleotide sequence whichis ligated to, or is manipulated to become ligated to, a nucleic acidsequence to which it is not ligated in nature, or to which it is ligatedat a different location in nature. Heterologous DNA is not endogenous tothe cell into which it is introduced, but has been obtained from anothercell. Heterologous DNA also includes an endogenous DNA sequence whichcontains some modification. Generally, although not necessarily,heterologous DNA encodes RNA and proteins that are not normally producedby the cell into which it is expressed. Examples of heterologous DNAinclude reporter genes, transcriptional and translational regulatorysequences, selectable marker proteins (e.g., proteins which confer drugresistance), etc.

The term “foreign gene” refers to any nucleic acid (e.g., gene sequence)which is introduced into the genome of a cell by experimentalmanipulations and may include gene sequences found in that cell so longas the introduced gene contains some modification (e.g., a pointmutation, the presence of a selectable marker gene, etc.) relative tothe naturally-occurring gene.

The term “transformation” as used herein refers to the introduction of atransgene into a cell. Transformation of a cell may be stable ortransient. The term “transient transformation” or “transientlytransformed” refers to the introduction of one or more transgenes into acell in the absence of integration of the transgene into the host cell'sgenome. Transient transformation may be detected by, for example,enzyme-linked immunosorbent assay (ELISA) which detects the presence ofa polypeptide encoded by one or more of the transgenes. Alternatively,transient transformation may be detected by detecting the activity ofthe protein (e.g., β-glucuronidase) encoded by the transgene (e.g., theuid A gene) as demonstrated herein [e.g., histochemical assay of GUSenzyme activity by staining with X-gluc which gives a blue precipitatein the presence of the GUS enzyme; and a chemiluminescent assay of GUSenzyme activity using the GUS-Light kit (Tropix)]. The term “transienttransformant” refers to a cell which has transiently incorporated one ormore transgenes. In contrast, the term “stable transformation” or“stably transformed” refers to the introduction and integration of oneor more transgenes into the genome of a cell. Stable transformation of acell may be detected by Southern blot hybridization of genomic DNA ofthe cell with nucleic acid sequences which are capable of binding to oneor more of the transgenes. Alternatively, stable transformation of acell may also be detected by the polymerase chain reaction of genomicDNA of the cell to amplify transgene sequences. The term “stabletransformant” refers to a cell which has stably integrated one or moretransgenes into the genomic DNA. Thus, a stable transformant isdistinguished from a transient transformant in that, whereas genomic DNAfrom the stable transformant contains one or more transgenes, genomicDNA from the transient transformant does not contain a transgene.

The term “nucleotide sequence of interest” refers to any nucleotidesequence, the manipulation of which may be deemed desirable for anyreason (e.g., confer improved qualities), by one of ordinary skill inthe art. Such nucleotide sequences include, but are not limited to,coding sequences of structural genes (e.g., reporter genes, selectionmarker genes, oncogenes, drug resistance genes, growth factors, etc.),and non-coding regulatory sequences which do not encode an mRNA orprotein product, (e.g., promoter sequence, polyadenylation sequence,termination sequence, enhancer sequence, etc.).

The term “isolated” when used in relation to a nucleic acid, as in “anisolated nucleic acid sequence” refers to a nucleic acid sequence thatis identified and separated from at least one contaminant nucleic acidwith which it is ordinarily associated in its natural source. Isolatednucleic acid is nucleic acid present in a form or setting that isdifferent from that in which it is found in nature. In contrast,non-isolated nucleic acids are nucleic acids such as DNA and RNA whichare found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs which encode a multitude of proteins. However,an isolated nucleic acid sequence comprising SEQ ID NO:7 includes, byway of example, such nucleic acid sequences in cells which ordinarilycontain SEQ ID NO:7 where the nucleic acid sequence is in a chromosomalor extrachromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid sequence may be present insingle-stranded or double-stranded form. When an isolated nucleic acidsequence is to be utilized to express a protein, the nucleic acidsequence will contain at a minimum at least a portion of the sense orcoding strand (i.e., the nucleic acid sequence may be single-stranded).Alternatively, it may contain both the sense and anti-sense strands(i.e., the nucleic acid sequence may be double-stranded).

As used herein, the term “purified” refers to molecules, either nucleicor amino acid sequences, that are removed from their naturalenvironment, isolated or separated. An “isolated nucleic acid sequence”is therefore a purified nucleic acid sequence. “Substantially purified”molecules are at least 60% free, preferably at least 75% free, and morepreferably at least 90% free from other components with which they arenaturally associated.

As used herein, the terms “complementary” or “complementarity” are usedin reference to nucleotide sequences related by the base-pairing rules.For example, the sequence 5′-AGT-3′ is complementary to the sequence5′-ACT-3′. Complementarity can be “partial” or “total.” “Partial”complementarity is where one or more nucleic acid bases is not matchedaccording to the base pairing rules. “Total” or “complete”complementarity between nucleic acids is where each and every nucleicacid base is matched with another base under the base pairing rules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands.

A “complement” of a nucleic acid sequence as used herein refers to anucleotide sequence whose nucleic acids show total complementarity tothe nucleic acids of the nucleic acid sequence.

The term “homology” when used in relation to nucleic acids refers to adegree of complementarity. There may be partial homology (i.e., partialidentity) or complete homology (i.e., complete identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or probe (i.e., an oligonucleotide which is capableof hybridizing to another oligonucleotide of interest) will compete forand inhibit the binding (i.e., the hybridization) of a completelyhomologous sequence to a target under conditions of low stringency. Thisis not to say that conditions of low stringency are such thatnon-specific binding is permitted; low stringency conditions requirethat the binding of two sequences to one another be a specific (i.e.,selective) interaction. The absence of non-specific binding may betested by the use of a second target which lacks even a partial degreeof complementarity (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described infra.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe which can hybridizeto the single-stranded nucleic acid sequence under conditions of lowstringency as described infra.

The term “hybridization” as used herein includes “any process by which astrand of nucleic acid joins with a complementary strand through basepairing.” [Coombs J (1994) Dictionary of Biotechnology, Stockton Press,New York N.Y.]. Hybridization and the strength of hybridization (i.e.,the strength of the association between the nucleic acids) is impactedby such factors as the degree of complementarity between the nucleicacids, stringency of the conditions involved, the T_(m) of the formedhybrid, and the G:C ratio within the nucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl [see e.g, Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985)]. Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of T_(m).

Low stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 68° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄·H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 1% SDS, 5×Denhardt's reagent [50× Denhardt's contains the following per 500 ml: 5g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 0.2×SSPE, and 0.1% SDS at room temperature when a DNA probeof about 100 to about 1000 nucleotides in length is employed.

High stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 68° C. in a solution consisting of 5×SSPE, 1% SDS, 5×Denhardt'sreagent and 100 μg/ml denatured salmon sperm DNA followed by washing ina solution comprising 0.1×SSPE, and 0.1% SDS at 68° C. when a probe ofabout 100 to about 1000 nucleotides in length is employed.

The term “equivalent” when made in reference to a hybridizationcondition as it relates to a hybridization condition of interest meansthat the hybridization condition and the hybridization condition ofinterest result in hybridization of nucleic acid sequences which havethe same range of percent (%) homology. For example, if a hybridizationcondition of interest results in hybridization of a first nucleic acidsequence with other nucleic acid sequences that have from 50% to 70%homology to the first nucleic acid sequence, then another hybridizationcondition is said to be equivalent to the hybridization condition ofinterest if this other hybridization condition also results inhybridization of the first nucleic acid sequence with the other nucleicacid sequences that have from 50% to 70% homology to the first nucleicacid sequence.

When used in reference to nucleic acid hybridization the art knows wellthat numerous equivalent conditions may be employed to comprise eitherlow or high stringency conditions; factors such as the length and nature(DNA, RNA, base composition) of the probe and nature of the target (DNA,RNA, base composition, present in solution or immobilized, etc.) and theconcentration of the salts and other components (e.g., the presence orabsence of formamide, dextran sulfate, polyethylene glycol) areconsidered and the hybridization solution may be varied to generateconditions of either low or high stringency hybridization differentfrom, but equivalent to, the above-listed conditions.

Those skilled in the art know that whereas higher stringencies may bepreferred to reduce or eliminate non-specific binding between thenucleotide sequence of SEQ ID NOs:7 and 10 and other nucleic acidsequences, lower stringencies may be preferred to detect a larger numberof nucleic acid sequences having different homologies to the nucleotidesequence of SEQ ID NOs:7 and 10.

The term “promoter,” “promoter element,” or “promoter sequence” as usedherein, refers to a DNA sequence which when ligated to a nucleotidesequence of interest is capable of controlling the transcription of thenucleotide sequence of interest into mRNA. A promoter is typically,though not necessarily, located 5′ (i.e., upstream) of a nucleotidesequence of interest whose transcription into mRNA it controls, andprovides a site for specific binding by RNA polymerase and othertranscription factors for initiation of transcription.

Promoters may be tissue specific or cell specific. The term “tissuespecific” as it applies to a promoter refers to a promoter that iscapable of directing selective expression of a nucleotide sequence ofinterest to a specific type of tissue (e.g., petals) in the relativeabsence of expression of the same nucleotide sequence of interest in adifferent type of tissue (e.g., roots). Tissue specificity of a promotermay be evaluated by, for example, operably linking a reporter gene tothe promoter sequence to generate a reporter construct, introducing thereporter construct into the genome of a plant such that the reporterconstruct is integrated into every tissue of the resulting transgenicplant, and detecting the expression of the reporter gene (e.g.,detecting mRNA, protein, or the activity of a protein encoded by thereporter gene) in different tissues of the transgenic plant. Thedetection of a greater level of expression of the reporter gene in oneor more tissues relative to the level of expression of the reporter genein other tissues shows that the promoter is specific for the tissues inwhich greater levels of expression are detected. The term “cell typespecific” as applied to a promoter refers to a promoter which is capableof directing selective expression of a nucleotide sequence of interestin a specific type of cell in the relative absence of expression of thesame nucleotide sequence of interest in a different type of cell withinthe same tissue. The term “cell type specific” when applied to apromoter also means a promoter capable of promoting selective expressionof a nucleotide sequence of interest in a region within a single tissue.Cell type specificity of a promoter may be assessed using methods wellknown in the art, e.g., eimmunohistochemical staining. Briefly, tissuesections are embedded in paraffin, and paraffin sections are reactedwith a primary antibody which is specific for the polypeptide productencoded by the nucleotide sequence of interest whose expression iscontrolled by the promoter. A labeled (e.g., peroxidase conjugated)secondary antibody which is specific for the primary antibody is allowedto bind to the sectioned tissue and specific binding detected (e.g.,with avidin/biotin) by microscopy.

Promoters may be constitutive or regulatable. The term “constitutive”when made in reference to a promoter means that the promoter is capableof directing transcription of an operably linked nucleic acid sequencein the absence of a stimulus (e.g., heat shock, chemicals, light, etc.).Typically, constitutive promoters are capable of directing expression ofa transgene in substantially any cell and any tissue. In contrast, a“regulatable” promoter is one which is capable of directing a level oftranscription of an operably linked nuclei acid sequence in the presenceof a stimulus (e.g, heat shock, chemicals, light, etc.) which isdifferent from the level of transcription of the operably linked nucleicacid sequence in the absence of the stimulus.

The terms “infecting” and “infection” with a bacterium refer toco-incubation of a target biological sample, (e.g., cell, tissue, etc.)with the bacterium under conditions such that nucleic acid sequencescontained within the bacterium are introduced into one or more cells ofthe target biological sample.

The term “Agrobacterium” refers to a soil-borne, Gram-negative,rod-shaped phytopathogenic bacterium which causes crown gall. The term“Agrobacterium” includes, but is not limited to, the strainsAgrobacterium tumefaciens, (which typically causes crown gall ininfected plants), and Agrobacterium rhizogens (which causes hairy rootdisease in infected host plants). Infection of a plant cell withAgrobacterium generally results in the production of opines (e.g.,nopaline, agropine, octopine etc.) by the infected cell. Thus,Agrobacterium strains which cause production of nopaline (e.g.' strainLBA4301, C58, A208) are referred to as “nopaline-type” Agrobacteria;Agrobacterium strains which cause production of octopine (e.g' strainLBA4404, Ach5, B6) are referred to as “octopine-type” Agrobacteria; andAgrobacterium strains which cause production of agropine (e.g., strainEHA105, EHA101, A281) are referred to as “agropine-type” Agrobacteria.

The terms “bombarding, “bombardment,” and “biolistic bombardment” referto the process of accelerating particles towards a target biologicalsample (e.g., cell, tissue, etc.) to effect wounding of the cellmembrane of a cell in the target biological sample and/or entry of theparticles into the target biological sample. Methods for biolisticbombardment are known in the art (e.g., U.S. Pat. No. 5,584,807, thecontents of which are herein incorporated by reference), and arecommercially available (e.g., the helium gas-driven microprojectileaccelerator (PDS-1000/He) (BioRad).

The term “microwounding” when made in reference to plant tissue refersto the introduction of microscopic wounds in that tissue. Microwoundingmay be achieved by, for example, particle bombardment as describedherein.

The term “plant” as used herein refers to a plurality of plant cellswhich are largely differentiated into a structure that is present at anystage of a plant's development. Such structures include, but are notlimited to, a fruit, shoot, stem, leaf, flower petal, etc. The term“plant tissue” includes differentiated and undifferentiated tissues ofplants including, but not limited to, roots, shoots, leaves, pollen,seeds, tumor tissue and various types of cells in culture (e.g., singlecells, protoplasts, embryos, callus, protocorm-like bodies, etc.). Planttissue may be in planta, in organ culture, tissue culture, or cellculture.

DESCRIPTION OF THE INVENTION

The present invention provides nucleic acid sequences having promoteractivity. The nucleic acid sequences provided herein direct constitutiveexpression of operably linked nucleotide sequences in cells, tissues andorgans of monocotyledonous and dicotyledonous plants. The promotersequences provided herein were discovered by screening highly expressedsugarcane polyubiquitin genes. The sequences of the invention arecapable of driving expression of operably linked nucleotide sequences inplant cells at a level which is comparable to or greater than thoselevels expressed under the control of the prior art's maizepolyubiquitin promoter sequences. Also provided by the invention aremethods for constitutive expression of a nucleic acid sequence ofinterest in plants. The nucleic acid sequences and methods of theinvention allow generation of transgenic plants which exhibitagronomically desirable characteristics.

The invention is further described under (A) Sugarcane PolyubiquitinPromoter Sequences, (B) Using Probes To Identify And Isolate Homologs ofThe Sugarcane Promoter Sequences, (C) Using Primers to AmplifyNucleotide Sequences, and (D) Generating Transgenic Plants.

A. Sugarcane Polyubiquitin Promoter Sequences

Ubiquitin involvement has been shown in protein turnover, heat shockresponse, and many other important cellular processes [Hershko et al.,Annual Review of Biochemistry 61, 761-808 (1992)]. Consistent with itsessential biological roles, the structure of the protein is very highlyconserved in all eukaryotes [Callis et al., Genetics 139, 921-39 (1995);Callis et al., Oxford Surv Plant Mol Cell Biol 6, 1-30 (1989); Sun etal., Plant J 11 , 1017-27 (1997)]. Many genes encoding ubiquitin containvarious numbers of tandem repeats of the entire protein coding regionand hence are called polyubiquitin genes. The primary translationproduct is a polyprotein, which is processed to form ubiquitin monomerspost-translationally.

Ubiquitin protein is abundant throughout the plant body, and severalpolyubiquitin genes have been shown to be expressed in most or all celltypes under most or all environmental conditions [Kawalleck et al.,Plant Mol Biol 21, 673-84 (1993)]. Numerous other polyubiquitin genes,however, are expressed in a tissue-specific manner [Callis et al., ProcNatl Acad Sci USA 91, 6074-7 (1994); Plesse et al., Mol Gen Genet 254,258-66 (1997)], or in response to environmental signals such as heatstress [Christensen et al., Plant Molecular Biology 12, 619-632 (1989);Liu et al., Biochem Cell Biol 73, 19-30 (1995)], or both [Almoguera etal., Plant Physiology 107, 765-773 (1995); Binet et al., Plant Mol Biol17, 395-407 (1991); Burke et al., Mol Gen Genet 213, 435-43 (1988);Garbarino et al., Plant Mol Biol 20, 235-44 (1992); Genschik et al.,Gene 148, 195-202 (1994); Sun et al. (1997) supra; Takimoto et al.,Plant Mol Biol 26, 1007-12 (1994)]. Several polyubiquitin promoters havebeen isolated and used to drive transgene expression [Christensen etal., Plant Mol Biol 18, 675-89 (1992); Garbarino et al., PlantPhysiology 109, 1371-1378 (1995)], including some which have been widelyused for constitutive expression of genes in plant transformation[Christensen et al., Transgenic Research 5, 213-218 (1996);Gallo-Meagher et al., Plant Cell Reporter 12, 666-670 (1993); Garbarinoet al. (1995) supra; Taylor et al., Plant Cell Reports 12, 491-495(1993); Quail et al., U.S. Pat. Nos. 5,614,399 and 5,510,474].

All Saccharum species are polyploid and most are at least octoploid(2N=40-128 or more). Commercial sugarcane cultivars are all Saccharumspecies hybrids derived from 2N+N chromosome transmission [Sreenivasanet al.: Cytogenics. In: Heinz DJ (ed) Sugarcane Improvement ThroughBreeding, pp. 211-253. Elsevier, Amsterdam (1987)]. This suggests thatsugarcane hybrids may have at least twelve alleles for each of themultiple genes that make up the polyubiquitin gene family.

The nucleic acid sequences of the invention were discovered during asearch by the inventors for promoters which are suitable for high-levelconstitutive transgene expression in monocotyledonous and dicotyledonousplants. The inventors isolated five polyubiquitin cDNA clones (scubi221,241, 511, 561, and 5121) from sugarcane stem tissue [Albert et al.,Plant Physiology 109, 337 (1995)]. Based on comparison of their 3′untranslated sequences, the inventors then grouped these five genes intofour “sub-families.” The inventors′ investigation of the expression oftwo members of these four sub-families and isolated genomic clones, ledto isolation of clones which contained promoters for two members of themost highly expressed sub-family.

The invention provides the nucleic acid sequence of two members of thesugarcane polyubiquitin family, the ubi4 gene and the ubi9 gene.Referring to the ubi4 gene, the initially determined nucleic acidsequence (SEQ ID NO:1) of the ubi4 gene including the translation startcodon (ATG) and the sequence upstream of the translation start codon isshown in FIG. 3A, and the nucleic acid sequence (SEQ ID NO:2) of theubi4 gene including the translation stop codon and sequences downstramof the translation stop codon is shown in FIG. 3B. The subsequentlydetermined nucleic acid sequence (SEQ ID NO:5) of the entire ubi4 geneis shown in FIG. 5A with the subsequently determined nucleic acidsequence (SEQ ID NO:7) located upstream of the translation start codonof the ubi4 gene being shown in FIG. 10. The nucleotide sequence of FIG.5A represents nucleotides 1 to 5512 of the 5551 nucleotide sequencedeposited as GenBank accession number AF093504.

Fragments of the ubi4 gene sequence which were identical in theinitially determined and the subsequently determined nucleic acidsequences were as follows (the nucleotide numbers refer to thenucleotide number in SEQ ID NO:5): Fragment A: 1-242; fragment B:245-787; fragment C: 788-1020; fragment D: 1021-1084; fragment E:1085-1168; fragment F: 1169-1173; fragment G: 1174-1648; and fragment H:1649-1805. The nucleotide sequence upstream of the transcription startcodon of the ubi4 gene (i.e., nucleotides 1-1810 of SEQ ID NO:1, andnucleotides 1-1802 of SEQ ID NOs:5 and 7) contained three regions: (a)an upstream of the 5′ UTR sequence (i.e., nucleotides 1-378 of SEQ IDNO:1, and nucleotides 1-377 of SEQ ID NOs:5 and 7),(b) a 5′ UTR sequence(i.e., nucleotides 379-444 of SEQ ID NO:1, and nucleotides 378-442 ofSEQ ID NOs:5 and 7); and (c) an intron sequence (i.e., nucleotides445-1810 of SEQ ID NO:1, and nucleotides 443-1802 of SEQ ID NOs:5 and7).

With respect of the ubi9 gene, the initially determined nucleic acidsequence (SEQ ID NO:3) of the ubi9 gene including the translation startcodon and sequences upstream thereof is shown in FIG. 7A and theinitially determined nucleic acid sequence (SEQ ID NO:4) of the ubi9gene including the translation stop codon and sequences downstreamthereof is shown in FIG. 7B. The subsequently determined nucleic acidsequence (SEQ ID NO:8) of the entire ubi9 gene is shown in FIG. 8A, withthe nucleic acid sequence (SEQ ID NO:10) of the ubi9 gene upstream ofthe translation start codon being shown in FIG. 11.

It is noted that while the 5′ end of the ubi9 gene was obtained bycleavage with HindIII which recognizes the sequence 5′-AAGCTT-3′,repeated sequencing of the 5′-end of the ubi9 gene showed instead thesequence 5′-AAGTTT-3′ (FIGS. 8 and 11). Thus, it is the inventors' viewthat the sequence of the full-length ubi9 gene (a) is as shown in FIG.8A (SEQ ID NO:8) (i.e., total number of nucleotides being 5174, with theten nucleotides at the 5′-end being 5′-AAGTTTTGnT-3′), (b) is as shownin FIG. 8A (SEQ ID NO:8) with the exception that it has a total numberof nucleotides of 5174, with the ten nucleotides at the 5′-end being5′-AAGCTTTGnT-3′, assuming substitution of the “T” at position 4 with a“C”), or (c) is as shown in FIG. 8A (SEQ ID NO:8) with the exceptionthat it has a total number of nucleotides of 5175, with the tennucleotides at the 5′-end being 5′-AAGCTTTTGn-3′, assuming insertion ofa “C” at position 4. Accordingly, any reference herein to SEQ ID NO:8 isintended to mean each and every one of the three nucleotide sequencesdescribed in the preceding sentence.

Similarly, it is the inventors' view that the sequence upstream of thetranslation start codon of the ubi9 gene (a) is as shown in FIG. 11 (SEQID NO:10) (i.e., total number of nucleotides being 3688, with the tennucleotides at the 5′-end being 5′-AAGTTTTGnT-3′), (b) is as shown inFIG. 11 (SEQ ID NO:10) with the exception that it has a total number ofnucleotides of 3688, with the ten nucleotides at the 5′-end being5′-AAGCTTTGnT-3′, assuming substitution of the “T” at position 4 with a“C”), or (c) is as shown in FIG. 11 (SEQ ID NO:10) with the exceptionthat it has a total number of nucleotides of 3689, with the tennucleotides at the 5′-end being 5′-AAGCTTTTGn-3′, assuming insertion ofa “° C.” at position 4. Accordingly, any reference herein to SEQ IDNO:10 is intended to mean each and every one of the three nucleotidesequences described in the preceding sentence.

Fragments of the ubi9 gene sequence which were identical in theinitially determined and the subsequently determined nucleic acidsequences were as follows (the nucleotide number refers to thenucleotide number in SEQ ID NO:8): Fragment A: 1-3600; fragment B:3602-3612; and fragment C: 3614-3691. The nucleotide sequence upstreamof the translation start codon of the ubi9 gene (i.e., nucleotides1-3691 of SEQ ID NO:3, and nucleotides 1-3691 of SEQ ID NO:8) containedthree regions: (a) an upstream of the 5′ UTR sequence (i.e., nucleotides1-2248 of SEQ ID NO:3, and nucleotides 1-2248 of SEQ ID NOs:8 and 10),(b) a 5′ UTR sequence (i.e., nucleotides 2249-2313 of SEQ ID NO:3, andnucleotides 2249-2313 of SEQ ID NOs:8 and 10), and (c) an intronsequence (i.e., nucleotides 2314-3688 of SEQ ID NO:3, and nucleotides2314-3688 of SEQ ID NOs:8 and 10). A BLAST search of the GenBankdatabase showed 100% homology to only a 20-22 bp region of nucleotides1-3688 of SEQ ID NOs:8 and 10 (i.e. the region of the ubi9 gene whichcontained the sequence upstream of the 5′ UTR, the 5′ UTR sequence, andthe intron sequence), 100% homology to only a 20 bp region ofnucleotides 1-2248 of SEQ ID NOs:8 and 10 (i.e., the sequence upstreamof the 5′ UTR), and 87% homology between a 135 bp fragment of thesequence upstream of the 5′ UTR of SEQ ID NOs:8 and 10 and a 118 bpfragment of Saccharum sp. glucose transporter mRNA, 3′ end (GenBankaccession number L21752).

Data presented herein demonstrates that plasmids which contain the uid Agene encoding β-glucuronidase (GUS) under the control of SEQ ID NO:7(which is equivalent to the initially determined SEQ ID NO:1) of theubi4 gene or under the control of SEQ ID NO:10 (which is equivalent tothe initially determined SEQ ID NO:3) of the ubi9 gene successfullydrive transient expression of GUS in monocotyledonous sugarcanesuspension cultured cells (Example 3), monocotyledonous sorghum callus(Example 5), monocotyledonous pineapple leaves, protocorm-like bodies,roots and fruit (Example 6), and in dicotyledonous tobacco leaves(Example 4), as well as stable expression in monocotyledonous sugar canecallus (Example 7), monocotyledonous rice callus (Example 8), anddicotyledonous tobacco leaves (Example 9).

The present invention is not limited to SEQ ID NO:7 but specificallycontemplates portions thereof. As used herein the term “portion” whenmade in reference to a nucleic acid sequence refers to a fragment ofthat sequence. The fragment may range in size from ten (10) contiguousnucleotide residues to the entire nucleic acid sequence minus onenucleic acid residue. Thus, a nucleic acid sequence comprising “at leasta portion of” a nucleotide sequence comprises from ten (10) contiguousnucleotide residues of the nucleotide sequence to the entire nucleotidesequence.

In a preferred embodiment, portions contemplated to be within the scopeof the invention include, but are not limited to, portions larger than20 nucleotide bases, more preferably larger than 100 nucleotide bases,the sequence upstream of the 5′ UTR sequence (i.e., nucleotide sequencefrom position 1 to 377 of SEQ ID NO:7), the 5′ UTR sequence (i.e.,nucleotides sequence from position 378 to 442 of SEQ ID NO:7), and theintron sequence (i.e., nucleotide sequence from position 443 to 1802 ofSEQ ID NO:7). In an alternative preferred embodiment, portions withinthe scope of the invention include portions larger than 20 nucleotidebases, more preferably larger than 100 nucleotide bases, those sequenceswhich are upstream of the translation start codon and which areidentical in the initially determined and subsequently determinedsequence of the ubi4 gene, and are exemplified by the nucleotidesequence from position 1 to 242, from position 245 to 787, from position788 to 1020, from position 1021 to 1084, from position 1085 to 1168,from position 1169 to 1173, from position 1174 to 1648, and fromposition 1649 to 1802 of SEQ ID NO:7. In yet another alternativepreferred embodiment, the portion contains the 377 bp sequence which isupstream of the 5′ UTR and which is highly homologous (>90% identity) inboth the ubi4 and ubi9 gene sequences, i.e., the nucleotide sequencefrom position 1 to 377 of SEQ ID NO:7.

It is contemplated that the present invention is not limited to SEQ IDNO:10 but specifically includes portions thereof. In a preferredembodiment, portions contemplated to be within the scope of theinvention include, but are not limited to, portions larger than 20nucleotide bases, more preferably larger than 100 nucleotide bases, thesequence upstream of the 5′ UTR sequence (i.e., nucleotide sequence fromposition 1 to 2248 of SEQ ID NO:10), the 5′ UTR sequence (i.e.,nucleotides sequence from position 2249 to 2313 of SEQ ID NO:10), andthe intron sequence (i.e., nucleotide sequence from position 2314 to3688 of SEQ ID NO:10). In an alternative preferred embodiment, portionswithin the scope of the invention include portions larger than 20nucleotide bases, more preferably larger than 100 nucleotide bases,those sequences which are upstream of the translation start codon andwhich are identical in the initially determined and subsequentlydetermined sequence of the ubi9 gene, and are exemplified by thenucleotide sequence from position 1 to 3600, from position 3602 to 3612,and from position 3614 to 3688 of SEQ ID NO:10. In yet anotheralternative preferred embodiment, the portion contains the sequencewhich is upstream of the transcription start codon and which is highlyhomologous (>90% identity) in both the ubi4 and ubi9 gene sequences,i.e., the nucleotide sequence from position 1671 to 2248 of SEQ IDNO:10. This sequence includes the MITE (from position 1706 to 1906)which is not homologous to sequences in the polyubiquitin ubi4 promoter.

The sequences of the present invention are not limited to SEQ ID NOs:7and 10 and portions thereof, but also include homologs of SEQ ID NOs:7and 10, as well as portions of these homologs. A nucleotide sequencewhich is a “homolog” of SEQ ID NOs:7 and 10 is defined herein as anucleotide sequence which exhibits greater than 61% identity (but not100% identity) to the sequence of SEQ ID NOs:7 and 10, respectively.

The present invention also contemplates functioning or functionalhomologs of SEQ ID NOs:7 and 10. A “functional homolog” of SEQ ID NOs:7and 10 is defined as a nucleotide sequence having less than 100%homology with SEQ ID NOs:7 and 10, respectively, and which has promoteractivity having some or all the characteristics (e.g., constitutivepromoter activity) of the promoter activity of SEQ ID NOs:7 and 10,respectively. Homologs of SEQ ID NOs:7 and 10, and of portions thereof,include, but are not limited to, nucleotide sequences having deletions,insertions or substitutions of different nucleotides or nucleotideanalogs as compared to SEQ ID NOs:7 and 10, respectively. Such homologsmay be produced using methods well known in the art.

The invention also contemplates at least a portion of SEQ ID NOs:7 and10, and homologs thereof having promoter activity. The term “promoteractivity” when made in reference to a nucleic acid sequence refers tothe ability of the nucleic acid sequence to initiate transcription of anoperably linked nucleotide sequence into mRNA. The terms “operablylinked,” “in operable combination,” and “in operable order” as usedherein refer to the linkage of nucleic acid sequences in a manner suchthat a nucleic acid molecule is capable of directing the transcriptionof nucleic acid sequence of interest and/or the synthesis of apolypeptide sequence of interest.

Promoter activity may be determined using methods known in the art. Forexample, a candidate nucleotide sequence whose promoter activity is tobe determined is ligated in-frame to a nucleic acid sequence of interest(e.g., a reporter gene sequence, a selectable marker gene sequence) togenerate a reporter vector, introducing the reporter vector into planttissue using methods described herein, and detecting the expression ofthe reporter gene (e.g., detecting the presence of encoded mRNA orencoded protein, or the activity of a protein encoded by the reportergene). The reporter gene may express a visible markers. Reporter genesystems which express visible markers include β-glucuronidase and itssubstrate (X-Gluc), luciferase and its substrate (luciferin), andβ-galactosidase and its substrate (X-Gal) which are widely used not onlyto identify transformants, but also to quantify the amount of transientor stable protein expression attributable to a specific vector system[Rhodes C A et al. (1995) Methods Mol Biol 55:121-131]. In a preferredembodiment, the reporter gene is a GUS gene. The selectable marker genemay confer antibiotic or herbicide resistance. Examples of reportergenes include, but are not limited to, dhfr which confers resistance tomethotrexate [Wigler M et al, (1980) Proc Natl Acad Sci 77:3567-70];npt, which confers resistance to the aminoglycosides neomycin and G-418[Colbere-Garapin F et al., (1981) J. Mol. Biol. 150:1-14] and als orpat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Detecting the presence of encoded mRNA orencoded protein, or the activity of a protein encoded by the reportergene or the selectable marker gene indicates that the candidatenucleotide sequence has promoter activity.

Sequences within a promoter which affect promoter activity may bedetermined by using deletion constructs such as those described bySherri et al. for the determination of HSP70 intron alterations whichimpact transcription of genes operably linked thereto [U.S. Pat. No.5,593,874, hereby incorporated by reference]. Briefly, severalexpression plasmids are constructed to contain a reporter gene under theregulatory control of different candidate nucleotide sequences which areobtained either by restriction enzyme deletion of internal sequences inSEQ ID NOs:7 and 10, restriction enzyme truncation of sequences at the5′ and/or 3′ end of SEQ ID NOs:7 and 10, or by the introduction ofsingle nucleic acid base changes by PCR into SEQ ID NOs:7 and 10.Expression of the reporter gene by the deletion constructs is detected.Detection of expression of the reporter gene in a given deletionconstruct indicates that the candidate nucleotide sequence in thatdeletion construct has promoter activity.

At the 3′ end of the nucleic acid sequence of interest, other DNAsequences may also be included, e.g., a 3′ untranslated regioncontaining a polyadenylation site and transcription termination sites.

The present invention is not limited to sense molecules of SEQ ID NOs:7and 10 but contemplates within its scope antisense molecules comprisinga nucleic acid sequence complementary to at least a portion (e.g., aportion greater than 100 nucletide bases in length and more preferablygreater than 200 nucleotide bases in length) of the nucleotide sequenceof SEQ ID NOs:7 and 10. These antisense molecules find use in, forexample, reducing or preventing expression of a gene whose expression iscontrolled by SEQ ID NOs:7 and 10.

The nucleotide sequence of SEQ ID NOs:7 and 10, portions, homologs andantisense sequences thereof may be synthesized by synthetic chemistrytechniques which are commercially available and well known in the art[see Caruthers MH et al., (1980) Nuc. Acids Res. Symp. Ser. 215-223;Horn T. et al., (1980) Nuc. Acids Res. Symp. Ser. 225-232].Additionally, fragments of SEQ ID NOs:7 and 10 can be made by treatmentof SEQ ID NOs:7 and 10 with restriction enzymes followed by purificationof the fragments by gel electrophoresis. Alternatively, sequences mayalso produced using the polymerase chain reaction (PCR) as described byMullis [U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, all of whichare hereby incorporated by reference]. SEQ ID NOs:7 and 10, portions,homologs and antisense sequences thereof may be ligated to each other orto heterologous nucleic acid sequences using methods well known in theart.

The nucleotide sequence of synthesized sequences may be confirmed usingcommercially available kits as well as using methods well known in theart which utilize enzymes such as the Klenow fragment of DNA polymeraseI, Sequenase®, Taq DNA polymerase, or thermostable T7 polymerase.Capillary electrophoresis may also be used to analyze the size andconfirm the nucleotide sequence of the products of nucleic acidsynthesis, restriction enzyme digestion or PCR amplification.

It is readily appreciated by those in the art that the sequences of thepresent invention may be used in a variety of ways. For example, thesesequences are useful in directing the expression of polypeptidesequences in vitro and in vivo. In plants, this is useful in determiningthe role of the polypeptide in disease development as well an inproducing transgenic plants with desirable agronomic characteristics asdescribed below. In addition, portions of the sequences of the inventioncan be used as probes for the detection and isolation of complementaryDNA sequences, and for the amplification of nucleotide sequences asdescribed below.

B. Using Probes to Identify and Isolate Homologs of the SugarcanePromoter Sequences

The invention provided herein is not limited to SEQ ID NO:7 and 10,homologs thereof, and portions thereof, having promoter activity, butincludes sequences having no promoter activity (i.e., non-functionalhomologs and non-functional portions of homologs). This may bedesirable, for example, where a portion of SEQ ID NOs:7 and 10 is usedas a probe to detect the presence of SEQ ID NOs:7 and 10, respectively,or of portions thereof in a sample.

As used herein, the term “probe” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, recombinantly or by PCR amplification, which is capableof hybridizing to a nucleotide sequence of interest. A probe may besingle-stranded or double-stranded. It is contemplated that any probeused in the present invention will be labelled with any “reportermolecule,” so that it is detectable in any detection system including,but not limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, calorimetric,gravimetric, magnetic, and luminescent systems. It is not intended thatthe present invention be limited to any particular detection system orlabel.

The probes provided herein are useful in the detection, identificationand isolation of, for example, sequences such as those listed as SEQ IDNOs:7 and 10 as well as of homologs thereof. Preferred probes are ofsufficient length (e.g., from about 9 nucleotides to about 20nucleotides or more in length) such that high stringency hybridizationmay be employed. In one embodiment, probes from 20 to 50 nucleotidebases in length are employed.

C. Using Primers to Amplify Nucleotide Sequences

The invention provided herein is not limited to SEQ ID NO:7 and 10,homologs thereof, and portions thereof, having promoter activity, butincludes sequences having no promoter activity. This may be desirable,for example, where a portion of the nucleic acid sequences set forth asSEQ ID NOs:7 and 10 is used as a primer for the amplification of nucleicacid sequences by, for example, polymerase chain reactions (PCR) orreverse transcription-polymerase chain reactions (RT-PCR). The term“amplification” is defined as the production of additional copies of anucleic acid sequence and is generally carried out using polymerasechain reaction technologies well known in the art [Dieffenbach CW and GSDveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring HarborPress, Plainview N.Y.]. As used herein, the term “polymerase chainreaction” (“PCR”) refers to the method of K. B. Mullis disclosed in U.S.Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, all of which are herebyincorporated by reference, which describe a method for increasing theconcentration of a segment of a target sequence in a mixture of genomicDNA without cloning or purification. This process for amplifying thetarget sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then annealed totheir complementary sequences within the target molecule. Followingannealing, the primers are extended with a polymerase so as to form anew pair of complementary strands. The steps of denaturation, primerannealing and polymerase extension can be repeated many times (i.e.,denaturation, annealing and extension constitute one “cycle”; there canbe numerous “cycles”) to obtain a high concentration of an amplifiedsegment of the desired target sequence. The length of the amplifiedsegment of the desired target sequence is determined by the relativepositions of the primers with respect to each other, and therefore, thislength is a controllable parameter. By virtue of the repeating aspect ofthe process, the method is referred to as the “polymerase chainreaction” (hereinafter “PCR”). Because the desired amplified segments ofthe target sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are the to be “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;and/or incorporation of ³²P-labeled deoxyribonucleotide triphosphates,such as dCTP or DATP, into the amplified segment). In addition togenomic DNA, any nucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process itself are, themselves, efficienttemplates for subsequent PCR amplifications. Amplified target sequencesmay be used to obtain segments of DNA (e.g., genes) for the constructionof targeting vectors, transgenes, etc.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long (e.g.,from about 9 nucleotides to about 20 nucleotides or more in length) toprime the synthesis of extension products in the presence of theinducing agent. Suitable lengths of the primers may be empiricallydetermined and depend on factors such as temperature, source of primerand the use of the method. In one embodiment, the present inventionemploys probes from 20 to 50 nucleotide bases in length.

The primers contemplated by the invention are useful in, for example,identifying sequences which are homologous to the sugarcane ubi4 andubi9 gene sequences in plants and in other organisms.

D. Generating Transgenic Plants

The present invention provides methods for constitutively expressing anucleotide sequence of interest in a cell, tissue, organ, and/ororganism. In one embodiment, the methods provided herein directconstitutive expression of a nucleotide sequence of interest inmonocotyledonous and dicotyledonous plant cells. In one embodiment, thisis accomplished by introducing into a plant cell a vector that containsa nucleotide sequence of interest operably linked to sequences providedherein which have promoter activity. The transformed plant cell isallowed to develop into a transgenic plant in which the nucleotidesequence of interest is preferably, though not necessarily, expressed insubstantially every tissue. These steps are further described below forspecific embodiments.

1. Expression Vectors For Plants

In one embodiment, the methods of the invention involve transformationof monocotyledonous tissue (sugarcane suspension cultured cells,sugarcane callus, rice callus, maize embryos, pineapple leaves,protocorm-like bodies, roots and fruit, and sorghum callus) anddicotyledonous tissue (tobacco leaves, tomato plants, and soybeanexcised embryonic meristems) with expression vectors in which theβ-glucuronidase (GUS) gene is under the transcriptional control of theexemplary sugarcane ubi4 gene promoter sequence (SEQ ID NO:7) or of theexemplary sugarcane ubi9 gene promoter sequence (SEQ ID NO: 10). As usedherein, the terms “vector” and “vehicle” are used interchangeably inreference to nucleic acid molecules that transfer DNA segment(s) fromone cell to another. The term “expression vector” as used herein refersto a recombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism.

The methods of the invention are not limited to the expression vectorsdisclosed herein. Any expression vector which is capable of introducinga nucleic acid sequence of interest into a plant cell is contemplated tobe within the scope of this invention. Typically, expression vectorscomprise the nucleic acid sequence of interest as well as companionsequences which allow the transcription of this sequence, and whichallow cloning of the vector into a bacterial or phage host. The vectorpreferably, though not necessarily, contains an origin of replicationwhich is functional in a broad range of prokaryotic hosts. A selectablemarker is generally, but not necessarily, included to allow selection ofcells bearing the desired vector.

In a preferred embodiment, the promoter sequence is SEQ ID NO:7 which isderived from the sugarcane ubi4 gene. In an alternative preferredembodiment, the promoter sequence is SEQ ID NO:10 which is derived fromthe sugarcane ubi9 gene. However, the invention is not limited to thepromoter sequences used herein. Any sequence which is a portion,homolog, or a homolog of a portion of SEQ ID NOs:7 and 10 and which haspromoter activity is contemplated to be within the scope of theinvention.

In addition to a promoter sequence, the expression vector preferablycontains a transcription termination sequence downstream of the nucleicacid sequence of interest to provide for efficient termination.Exemplary termination sequences include the nopaline synthase (NOS)termination sequence, and different fragments of the sugarcaneribulose-1,5-biphosphate carboxylase/oxygenase (rubisco) small subunit(scrbcs) gene. The termination sequences of the expression vectors arenot critical to the invention. The termination sequence may be obtainedfrom the same gene as the promoter sequence or may be obtained fromdifferent genes.

If the mRNA encoded by the nucleic acid sequence of interest is to beefficiently translated, polyadenylation sequences are also commonlyadded to the expression vector. Examples of the polyadenylationsequences include, but are not limited to, the Agrobacterium octopinesynthase signal, or the nopaline synthase signal. Where it is preferredthat the nucleic acid sequence of interest is not translated into apolypeptide (e.g., where the nucleic acid sequence of interest encodesan antisense RNA), polyadenylation signals are not necessary.

Vectors for the transformation of plant cells are not limited to thetype or nature of the expressed genes disclosed herein. Any nucleic acidsequence of interest may be used to create transgenic plant cells,tissues, organs, and plants. Nucleic acid sequences of interest includesequences which encode a protein of interest. The terms “protein ofinterest” and “polypeptide of interest” refer to any protein orpolypeptide, respectively, the manipulation of which may be deemeddesirable for any reason, by one of ordinary skill in the art.

For example, it may be desirable to express a nucleic acid sequencewhich encodes a polypeptide sequence having, for example, enzymeactivity. One example of such an enzyme is theI-arninocyclopropane-l-carboxylic acid (ACC) deaminase enzyme whichmetabolizes ACC in plant tissue thereby lowering the level of ethylenewhich is responsible for fruit ripening (U.S. Pat. No. 5,512,466, thecontents of which are hereby incorporated by reference).

Another enzyme which may be desirably expressed in a plant is thesucrose phosphate synthase enzyme which increases the level of sucrosein the fruit. The nucleic acid sequence of the gene encoding this enzymeis known [e.g., in maize; Worrell et al. (1991) Plant Cell 3:1121-1130](FIG. 15) (SEQ ID NO:12) and has been assigned GenBank accession numberm97550.

Yet another example of a suitable enzyme for use in this invention isEPSP synthase (5-enolpyruvyl-3- phosphoshikimate synthase; EC:25.1.19)which is an enzyme involved in the shikimic acid pathway of plants. Theshikimic acid pathway provides a precursor for the synthesis of aromaticamino acids essential to the plant. Specifically, EPSP synthasecatalyzes the conversion of phosphoenol pyruvate and 3-phosphoshikimicacid to 5-enolpyruvyl-3- phosphoshikimate acid. A herbicide containingN-phosphonomethylglycine inhibits the EPSP synthase enzyme and therebyinhibits the shikimic acid pathway of the plant. The term “glyphosate”is usually used to refer to the N-phosphonomethylglycine herbicide inits acidic or anionic forms. Novel EPSP synthase enzymes have beendiscovered that exhibit an increased tolerance to glyphosate containingherbicides. In particular, an EPSP synthase enzyme having a singleglycine to alanine substitution in the highly conserved region havingthe sequence: -L-G-N-A-G-T-A- located between positions 80 and 120 inthe mature wild-type EPSP synthase amino add sequence has been shown toexhibit an increased tolerance to glyphosate and is described in U.S.Patent No. 4,971,908, the teachings of which are hereby incorporated byreference. Methods for transforming plants to exhibit glyphosatetolerance are discussed in U.S. Pat. No. 4,940,835, incorporated hereinby reference. A glyphosate-tolerant EPSP synthase plant gene encodes apolypeptide which contains a chloroplast transit peptide (CTP) whichenables the EPSP synthase polypeptide (or an active portion thereto) tobe transported into a chloroplast inside the plant cell. The EPSPsynthase gene is transcribed into mRNA in the nucleus and the mRNA istranslated into a precursor polypeptide (CTP/mature EPSP synthase) inthe cytoplasm. The precursor polypeptide is transported into thechloroplast.

Additional examples of enzymes suitable for use in this invention areacetolactate synthase, RNase to impart male sterility [Mariani et al.(1990) Nature 347: 737-741], and wheat germ agglutinin.

Yet other examples of desirable nucleic acid sequence are those whichencode the sweetness protein. The nucleic acid sequence for the geneencoding the sweetness protein is known in the art (see, e.g., U.S.patent application Ser. No. 08/670,186, the contents of which are hereinincorporated by reference). Transformation of plants with the sweetnessprotein is useful in, for example, providing a base level of sweetnessin the fruit, thus reducing the effects of differences in fruit maturityby providing more uniform sweetness in different parts of the fruit.

Further examples of suitable proteins for use in this invention areBacillus thuringiensis (B.t.) crystal toxin proteins which whenexpressed in plants protect the plants from insect infestation becausethe insect, upon eating the plant containing the B.t. toxin proteineither dies or stops feeding. B.t. toxin proteins which are toxic toeither Lepidopteran or Coleopteran insects may be used. Examples ofparticularly suitable DNA sequences encoding B.t. toxin protein aredescribed in the EP patent application Ser. No.385,962 entitled“Synthetic Plant Genes and Method for Preparation,” published Sep. 5,1990.

Alternatively, it may be desirable to express a nucleic acid sequencewhich encodes an antisense RNA that hybridizes with a genomic plant DNAsequence. For example, it may be of advantage to express antisense RNAwhich is specific for genomic plant DNA sequences that encode an enzymewhose activity is sought to be decreased. Examples of DNA sequenceswhose reduced expression may be desirable are known in the artincluding, but not limited to, the ethylene inducible sequences in fruit(U.S. Pat. No. 5,545,815, the entire contents of which are hereinincorporated by reference). Expression of antisense RNA which ishomologous with these ethylene inducible sequences is useful in delayingfruit ripening and in increasing fruit firmness. Other DNA sequenceswhose expression may be desirably reduced include the ACC synthase genewhich encodes the ACC synthase enzyme that is the first and ratelimiting step in ethylene biosynthesis. Nucleic acid sequences for thisgene have been described from a number of plant sources (e.g., Picton etal. (1993) The Plant J. 3:469-481; U.S. Pat. Nos. 5,365,015 and5,723,766, the contents of both of which are herein incorporated byreference). Expression of antisense RNA which hybridizes with ACCsynthase genomic sequences in plants may be desirable to delay fruitripening.

Yet another sequence whose expression may be advantageously reduced isthe genomic sequence encoding the enzyme polyphenol oxidase. This enzymeis involved in the browning reaction that occurs during chilling injury.Nucleic acid sequences encoding this enzyme have been previouslydescribed in the art (e.g., Shahar et al. (1992) Plant Cell 4:135-147],as shown in FIG. 14 (SEQ ID NO:11) (GenBank accession number s40548).The use of antisense polyphenol antisense sequences has been reported toinhibit polyphenol oxidase (PPO) gene expression and to inhibit browning[Bachem et al. (1994) Bio/Technology 12:1101-1105].

One of skill in the art knows that the antisense DNA segment to beintroduced into the plant may include the fall length coding region ofthe targeted gene or a portion thereof. Complete homology between thenucleotide sequences of the antisense RNA and the targeted genomic DNAis not required. Rather, antisense DNA sequences which encode antisenseRNA sequences that are partially homologous to a targeted genomic DNAsequence are contemplated to be within the scope of the invention solong as the antisense RNA sequences are capable of repressing expressionof the target genomic DNA sequence.

The invention is not limited to vectors which express a single nucleicacid sequence of interest. Vectors which contain a plurality of (i.e.,two or more) nucleic acid sequences under the transcriptional control ofthe same promoter sequence are expressly contemplated to be within thescope of the invention. Such vectors may be desirable, for example,where the expression products of the plurality of nucleic acid sequencescontained within the vector provide protection against differentpathogens, and where simultaneous protection against these differentpathogens is deemed advantageous.

Also included within the scope of this invention are vectors whichcontain the same or different nucleic acid sequences under thetranscriptional control of different promoter sequences derived from SEQID NO:7, SEQ ID NO:10, and other sequences. Such vectors may bedesirable to, for example, to control different levels of expression ofdifferent nucleic acid sequences of interest in plant tissues.

2. Transformation of Plant Cells

Once an expression vector is prepared, transgenic plants and plant cellsare obtained by introducing the expression vectors into plants and plantcells using methods known in the art. The present invention is suitablefor any member of the monocotyledonous (monocot) plant family including,but not limited to, maize, rice, barley, oats, wheat, sorghum, rye,sugarcane, pineapple, yams, onion, banana, coconut, dates and hops. Thepresent invention is also suitable for any member of the dicotyledonous(dicot) plant family including, but not limited to, tobacco, tomato,soybean, and papaya.

In one embodiment, the expression vectors are introduced into plantcells by particle mediated gene transfer. Particle mediated genetransfer methods are known in the art, are commercially available, andinclude, but are not limited to, the gas driven gene delivery instrumentdescried in McCabe, U.S. Pat. No. 5,584,807, the entire contents ofwhich are herein incorporated by reference. This method involves coatingthe nucleic acid sequence of interest onto heavy metal particles, andaccelerating the coated particles under the pressure of compressed gasfor delivery to the target tissue.

Other particle bombardment methods are also available for theintroduction of heterologous nucleic acid sequences into plant cells.Generally, these methods involve depositing the nucleic acid sequence ofinterest upon the surface of small, dense particles of a material suchas gold, platinum, or tungsten. The coated particles are themselves thencoated onto either a rigid surface, such as a metal plate, or onto acarrier sheet made of a fragile material such as mylar. The coated sheetis then accelerated toward the target biological tissue. The use of theflat sheet generates a uniform spread of accelerated particles whichmaximizes the number of cells receiving particles under uniformconditions, resulting in the introduction of the nucleic acid sampleinto the target tissue.

Alternatively, an expression vector may be inserted into the genome ofplant cells by infecting the cells with a bacterium, including but notlimited to an Agrobacterium strain previously transformed with thenucleic acid sequence of interest. Since most dicotyledonous plant arenatural hosts for Agrobacterium, almost every dicotyledonous plant maybe transformed by Agrobacterium in vitro. Although monocotyledonousplants, and in particular, cereals and grasses, are not natural hosts toAgrobacterium, work to transform them using Agrobacterium has also beencarried out (Hooykas-Van Slogteren et al., (1984) Nature 311:763-764).Plant genera that may be transformed by Agrobacterium includeChrysanthemum, Dianthus, Gerbera, Euphorbia. Pelaronium, Ipomoea,Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalum,Allium, Lilium, Narcissus, Ananas, Arachis, Phaseolus and Pisum.

For transformation with Agrobacterium, disarmed Agrobacterium cells aretransformed with recombinant Ti plasmids of Agrobacterium tumefaciens orRi plasmids of Agrobacterium rhizogenes (such as those described in U.S.Pat. No. 4,940,838, the entire contents of which are herein incorporatedby reference) which are constructed to contain the nucleic acid sequenceof interest using methods well known in the art [J. Sambrook et al(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,NY]. The nucleic acid sequence of interest is then stably integratedinto the plant genome by infection with the transformed Agrobacteriumstrain. For example, heterologous nucleic acid sequences have beenintroduced into plant tissues using the natural DNA transfer system ofAgrobacterium tumefaciens and Agrobacterium rhizogenes bacteria (forreview, see Klee et al. (1987) Ann. Rev. Plant Phys. 38:467-486).

Construction of recombinant Ti and Ri plasmids in general followsmethods typically used with the more common bacterial vectors, such aspBR322. Additional use can be made of accessory genetic elementssometimes found with the native plasmids and sometimes constructed fromforeign sequences. These may include but are not limited to structuralgenes for antibiotic resistance as selection genes.

There are two systems of recombinant Ti and Ri plasmid vector systemsnow in use. The first system is called the “cointegrate” system. In thissystem, the shuttle vector containing the gene of interest is insertedby genetic recombination into a non-oncogenic Ti plasmid that containsboth the cis-acting and trans-acting elements required for planttransformation as, for example, in the pMLJ1 shuttle vector and thenon-oncogenic Ti plasmid pGV3850. The second system is called the“binary” system in which two plasmids are used; the gene of interest isinserted into a shuttle vector containing the cis-acting elementsrequired for plant transformation. The other necessary functions areprovided in trans by the non-oncogenic Ti plasmid as exemplified by thepBIN19 shuttle vector and the non-oncogenic Ti plasmid PAL4404. Some ofthese vectors are commercially available.

There are three common methods to transform plant cells withAgrobacterium: The first method is by co-cultivation of Agrobacteriumwith cultured isolated protoplasts. This method requires an establishedculture system that allows culturing protoplasts and plant regenerationfrom cultured protoplasts. The second method is by transformation ofcells or tissues with Agrobacterium. This method requires (a) that theplant cells or tissues can be transformed by Agrobacterium and (b) thatthe transformed cells or tissues can be induced to regenerate into wholeplants. The third method is by transformation of seeds, apices ormeristems with Agrobacterium. This method requires micropropagation.

One of skill in the art knows that the efficiency of transformation byAgrobacterium may be enhanced by using a number of methods known in theart. For example, the inclusion of a natural wound response moleculesuch as acetosyringone (AS) to the Agrobacterium culture has been shownto enhance transformation efficiency with Agrobacterium tumefaciens[Shahla et al. (1987) Plant Molec. Biol. 8:291-298]. Alternatively,transformation efficiency may be enhanced by wounding the target tissueto be transformed. Wounding of plant tissue may be achieved, forexample, by punching, maceration, bombardment with microprojectiles,etc. [see, e.g., Bidney et al. (1992) Plant Molec. Biol. 18:301-313].

It may be desirable to target the nucleic acid sequence of interest to aparticular locus on the plant genome. Site-directed integration of thenucleic acid sequence of interest into the plant cell genome may beachieved by, for example, homologous recombination usingAgrobacterium-derived sequences. Generally, plant cells are incubatedwith a strain of Agrobacterium which contains a targeting vector inwhich sequences that are homologous to a DNA sequence inside the targetlocus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, aspreviously described (Offringa et al., (1996), U.S. Pat. No. 5,501,967,the entire contents of which are herein incorporated by reference). Oneof skill in the art knows that homologous recombination may be achievedusing targeting vectors which contain sequences that are homologous toany part of the targeted plant gene, whether belonging to the regulatoryelements of the gene, or the coding regions of the gene. Homologousrecombination may be achieved at any region of a plant gene so long asthe nucleic acid sequence of regions flanking the site to be targeted isknown.

Where homologous recombination is desired, the targeting vector used maybe of the replacement- Or insertion-type (Offringa et al. (1996),supra). Replacement-type vectors generally contain two regions which arehomologous with the targeted genomic sequence and which flank aheterologous nucleic acid sequence, e.g., a selectable marker genesequence. Replacement-type vectors result in the insertion of theselectable marker gene which thereby disrupts the targeted gene.Insertion-type vectors contain a single region of homology with thetargeted gene and result in the insertion of the entire targeting vectorinto the targeted gene.

Other methods are also available for the introduction of expressionvectors into plant tissue, e.g., electroinjection (Nan et al. (1995) In“Biotechnology in Agriculture and Forestry,” Ed. Y.P.S. Bajaj,Springer-Verlag Berlin Heidelberg, Vol 34:145-155; Griesbach (1992)HortScience 27:620); fusion with liposomes, lysosomes, cells, minicellsor other fusible lipid-surfaced bodies (Fraley et al. (1982) Proc. Natl.Acad. Sci. USA 79:1859-1863); polyethylene glycol (Krens et al. (1982)nature 296:72-74); chemicals that increase free DNA uptake;transformation using virus, and the like.

3. Selection of Transgenic Plant Cells

Plants, plant cells and tissues transformed with a heterologous nucleicacid sequence of interest are readily detected using methods known inthe art including, but not limited to, restriction mapping of thegenomic DNA, PCR-analysis, DNA-DNA hybridization, DNA-RNA hybridization,DNA sequence analysis and the like.

Additionally, selection of transformed plant cells may be accomplishedusing a selection marker gene. It is preferred, though not necessary,that a selection marker gene be used to select transformed plant cells.A selection marker gene may confer positive or negative selection.

A positive selection marker gene may be used in constructs for randomintegration and site-directed integration. Positive selection markergenes include antibiotic resistance genes, and herbicide resistancegenes and the like. In one embodiment, the positive selection markergene is the NPTII gene which confers resistance to geneticin (G418) orkanamycin. In another embodiment the positive selection marker gene isthe HPT gene which confers resistance to hygromycin. The choice of thepositive selection marker gene is not critical to the invention as longas it encodes a functional polypeptide product. Positive selection genesknown in the art include, but are not limited to, the ALS gene(chlorsulphuron resistance), and the DHFR-gene (methothrexateresistance).

A negative selection marker gene may also be included in the constructs.The use of one or more negative selection marker genes in combinationwith a positive selection marker gene is preferred in constructs usedfor homologous recombination. Negative selection marker genes aregenerally placed outside the regions involved in the homologousrecombination event. The negative selection marker gene serves toprovide a disadvantage (preferably lethality) to cells that haveintegrated these genes into their genome in an expressible manner. Cellsin which the targeting vectors for homologous recombination are randomlyintegrated in the genome will be harmed or killed due to the presence ofthe negative selection marker gene. Where a positive selection markergene is included in the construct, only those cells having the positiveselection marker gene integrated in their genome will survive.

The choice of the negative selection marker gene is not critical to theinvention as long as it encodes a functional polypeptide in thetransformed plant cell. The negative selection gene may for instance bechosen from the aux-2 gene from the Ti-plasmid of Agrobacterium, thetk-gene from SV40, cytochrome P450 from Streptomyces griseolus, theAdh-gene from Maize or Arabidopsis, etc. Any gene encoding an enzymecapable of converting a substance which is otherwise harmless to plantcells into a substance which is harmful to plant cells may be used.

4. Regeneration of Transgenic Plants

The present invention provides transgenic plants. The transgenic plantsof the invention are not limited to plants in which each and every cellexpresses the nucleic acid sequence of interest under the control of thesequences provided herein. Included within the scope of this inventionis any plant which contains at least one cell which expresses thenucleic acid sequence of interest (e.g., chimeric plants). It ispreferred, though not necessary, that the transgenic plant express thenucleic acid sequence of interest in more than one cell, and morepreferably in one or more tissue.

Once transgenic plant tissue which contains an expression vector hasbeen obtained, transgenic plants may be regenerated from this transgenicplant tissue using methods known in the art. The term “regeneration” asused herein, means growing a whole plant from a plant cell, a group ofplant cells, a plant part or a plant piece (e.g., from a protoplast,callus, protocorm-like body, or tissue part).

Species from the following examples of genera of plants may beregenerated from transformed protoplasts: Fragaria, Lotus, Medicago,Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium,Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa,Capsicum, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,Digitalis, Majorana, Ciohorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium,Zea, Triticum, Sorghum, and Datura.

For regeneration of transgenic plants from transgenic protoplasts, asuspension of transformed protoplasts or a petri plate containingtransformed explants is first provided. Callus tissue is formed andshoots may be induced from callus and subsequently rooted.Alternatively, somatic embryo formation can be induced in the callustissue. These somatic embryos germinate as natural embryos to formplants. The culture media will generally contain various amino acids andplant hormones, such as auxin and cytokinins. It is also advantageous toadd glutamic acid and proline to the medium, especially for such speciesas corn and alfalfa. Efficient regeneration will depend on the medium,on the genotype, and on the history of the culture. These threevariables may be empirically controlled to result in reproducibleregeneration.

Plants may also be regenerated from cultured cells or tissues.Dicotyledonous plants which have been shown capable of regeneration fromtransformed individual cells to obtain transgenic whole plants include,for example, apple (Malus pumila), blackberry (Rubus),Blackberry/raspberry hybrid (Rubus), red raspberry (Rubus), carrot(Daucus carota), cauliflower (Brassica oleracea), celery (Apiumgraveolens), cucumber (Cucumis sativus), eggplant (Solanum melongena),lettuce (Lactuca sativa), potato (Solanum tuberosum), rape (Brassicanapus), wild soybean (Glycine canescens), strawberry (Fragaria xananassa), tomato (Lycopersicon esculentum), walnut (Juglans regia),melon (Cucumis melo), grape (Vitis vinifera), and mango (Mangiferaindica). Monocotyledonous plants which have been shown capable ofregeneration from transformed individual cells to obtain transgenicwhole plants include, for example, rice (Oryza sativa), rye (Secalecereale), and maize.

In addition, regeneration of whole plants from cells (not necessarilytransformed) has also been observed in: apricot (Prunus armeniaca),asparagus (Asparagus officinalis), banana (hybrid Musa), bean (Phaseolusvulgaris), cherry (hybrid Prunus), grape (Vitis vinifera), mango(Mangifera indica), melon (Cucumis melo), ochra (Abelmoschusesculentus), onion (hybrid Allium), orange (Citrus sinensis), papaya(Carrica papaya), peach (Prunus persica), plum (Prunus domestica), pear(Pyrus communis), pineapple (Ananas comosus), watermelon (Citrullusvulgaris), and wheat (Triticum aestivun).

The regenerated plants are transferred to standard soil conditions andcultivated in a conventional manner. After the expression vector isstably incorporated into regenerated transgenic plants, it can betransferred to other plants by vegetative propagation or by sexualcrossing. For example, in vegetatively propagated crops, the maturetransgenic plants are propagated by the taking of cuttings or by tissueculture techniques to produce multiple identical plants. In seedpropagated crops, the mature transgenic plants are self crossed toproduce a homozygous inbred plant which is capable of passing thetransgene to its progeny by Mendelian inheritance. The inbred plantproduces seed containing the nucleic acid sequence of interest. Theseseeds can be grown to produce plants that would produce the selectedphenotype. The inbred plants can also be used to develop new hybrids bycrossing the inbred plant with another inbred plant to produce a hybrid.

Confirmation of the transgenic nature of the cells, tissues, and plantsmay be performed by PCR analysis, antibiotic or herbicide resistance,enzymatic analysis and/or Southern blots to verify transformation.Progeny of the regenerated plants may be obtained and analyzed to verifywhether the transgenes are heritable. Heritability of the transgene isfurther confirmation of the stable transformation of the transgene inthe plant.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

EXAMPLE 1 Isolation and Characterization of Sugarcane Polyubiquitin ubi4And ubi9 Genes

A. Plant materials

The plant materials used for the cDNA library and for expression studieswere sugarcane hybrid H65-7052 plants grown in the greenhouse of theHawaii Agriculture Research Center, Aiea, Hawaii. The genomic libraryused to isolate the genomic clones was made from the related sugarcanehybrid H32-8560 [Albert et al., Plant Mol Biol 20, 663-71 (1992)].Internodes were numbered consecutively down the culm, with number onedefined as that internode subtending the youngest fully expanded leaf,as previously described [Moore P. H.: Anatomy and Morphology. In: HeinzDJ (ed) Sugarcane Improvement Through Breeding, pp. 85-142. Elsevier,Amsterdam (1987)].

Sequence comparisons of the 3′ untranslated regions (UTR) of fivepolyubiquitin cDNA clones revealed significant differences, except forclones scubi241 and 511, which were identical in both 5′ and 3′ UTRsequences. Despite these identical regions, these clones do notrepresent transcripts from the same gene, as scubi241 contained fourcopies of the polyubiquitin coding repeat, whereas scubi511 containedfive of these repeats (data not shown).

B. Plasmid DNA gel blots

Polyubiquitin cDNA plasmid clones were digested with appropriaterestriction enzymes and size fractionated by gel electrophoresis. DNAwas transferred to Hybond-N+ (Amersham) membranes by capillary transfer.Identical DNA gel blots of the five clones were hybridized to 3′ UTRprobes at high stringency. High stringency hybridization and stringencywashes at 65° C. were by the method of [Church et al., Proc. Natl. Acad.Sci. USA 81, 1991-1995 (1984)]. Probe templates were prepared by PCRamplification of the 3′ UTR of each cDNA clone. Probes were ³²P labeledby the method of [Feinberg et al., Anal. Biochem. 132, 6-13 (1983)].Blots were exposed to Kodak X-Omat RP XRP-5 film for five to 10 min atroom temperature.

C. RNA extraction

Total RNA was extracted from 10 g of each tissue sample by the method of[Bugos et al., Biotechniques 19, 734-737 (1995)]. RNA concentration wasdetermined by spectrophotometer. Poly-A+ RNA was isolated from total RNAusing the PolyATract System (Promega).

D. RNA gel blot analysis

Fifteen μg of each RNA sample was separated by denaturing gelelectrophoresis as described by [Fourney et al., Focus 10, 5-7 (1988)].Hybridization and stringency washes at 65° C. were by the simplifiednorthern blot method of [Virca et al., Bio Techniques 8, 370-371(1990)]. ³²P incorporation into probes was monitored by two methods:scintillation counting following Sephadex G-50 chromatography and TCAprecipitation. Equal activity (1.7×10⁷ cpm/ml) of ³²P-labeledgene-specific probe was used for each hybridization. Autoradiography waswith Kodak X-Omat RP XRP-5 film that was preflashed to an OD₅₄₀ of 0.15to maximize linearity of response, exposed at −80° C.

Four identical RNA gel blots, each containing 15 μg total RNA frommature leaves, immature leaves, stem apices, internode 1, internode 2,internode 3, roots, and callus cultures at control (26° C.) and heatshock (37° C.) temperatures, were hybridized to each of the four genespecific 3′ UTR probes, respectively, and exposed to film for 16 h asshown in FIG. 1.

In FIG. 1, 15 μg total RNA from each indicated tissue was hybridized toequal activities of each gene-specific probe. Ethidium bromide stainedgel indicates equal loading of RNA from each tissue. M.L., matureleaves; I.L., immature leaves; S.A., shoot apex; I1, internode 1; I2,internode 2; I3, internode 3; R, roots; C, callus 26° C.; C-HS, callus37° C. Autoradiogram for scubi221 probe was exposed for 72 h, all othersfor 16 h. With this exposure time of 16 h, transcripts homologous toscubi221 were not detected except in callus tissue under 37° C.heat-shock, where a single band was barely detectable. After 72 hexposure, the single band in heat-shock callus was clear, but no clearsignal was evident for any other tissue. The scubi241/511 probe detectedtranscripts of two size classes (on the 16 h autoradiogram exposureshown in FIG. 1 the two bands overlap, however on shorter exposures twobands can be resolved), with both transcripts present in approximatelyequal amounts in all tested tissues. Transcripts for this sub-familywere present at the highest levels of all tested polyubiquitin genes;heat shock at 37° C. did not induce a significant change in transcriptaccumulation. The scubi561 probe also detected two mRNA size classes;however, the levels of these transcripts were considerably lower thanthose detected with the 241/511 probe. Transcript pools hybridizing tothe 561 probe were elevated significantly by 37° C. heat shock. Thescubi5121 probe hybridized to a single size class that was moderatelyabundant, but significantly lower than the scubi241/511 pool. The 5121levels were very similar in all tissues except callus. Callus at controltemperature (26° C.) contained less scubi5121 mRNA than did othertissues; 37° C. heat shock elevated transcript levels in callus tolevels approximately equal to those in other tissues at 26° C. Thispattern of reduced transcript levels in control temperature callustissue could be seen to some extent for all probes except scubi561.

To confirm the northern analysis results, a “reverse northern” blotcontaining the 3′ UTR DNA of all five polyubiquitin cDNA clones washybridized to a 32P-labeled, first strand cDNA probe made fromapproximately 0.5 μg mRNA extracted from immature leaves. Fifty ng ofeach 3′ UTR target DNA was loaded for the blot, ensuring that the targetDNA would be present in excess and that hybridization signals shouldreflect relative abundance of the different mRNAs in the template mRNApopulation. Results of this experiment are shown in FIG. 2.

In FIG. 2, 50 ng DNA from the gene-specific 3′ UTR of each cDNA clonewas hybridized to a total first strand cDNA probe from immature leaves.Results of this experiment confirmed the northern results, withscubi241/511 transcripts most abundant, scubi221 least abundant, andscubi561 and 5121 at intermediate levels (FIG. 2). Because these twoindependent methods of estimating the relative mRNA abundance for thedifferent polyubiquitin gene sub-families both indicated thescubi241/511 sub-family as highest, and because the difference betweenscubi241/511 and the other sub-families was so large by both measures,this ranking of expression levels is accurate.

While expression of these sugarcane polyubiquitin genes was littleaffected by cell-type, several of them responded dramatically to anenvironmental stimulus: heat stress. mRNA pools homologous to scubi221,561 and 5121 in sugarcane callus tissue subjected to 37° C. heat shockall rose substantially, whereas the pool homologous to scubi241/511 didnot show a substantial increase. Overall, scubi241 and scubi511 mostnearly fit the stereotype of “constitutive” genes, with uniformly highlevels of mRNA accumulation in all tested tissues and little inductionof expression by heat stress.

Approximately 1×10⁶ pfu from a sugarcane genomic library in the vectorλEMBL4 were screened with a polyubiquitin coding sequence probe. Fiftypolyubiquitin positive plaques were screened again and purified with thescubi241/511 specific probe. Five scubi241/511 positive plaques wereisolated, and two of these, λubi4 and λubi9, were subcloned into plasmidvectors for further analysis and sequencing.

Subclone pubi4 sequence and organization were initially determined to beas shown in FIGS. 3A, 3B and 4. Tables 1-2 provide the meaning ofsequence symbols other than the bases G, A, T, and C which indicatesambiguities where two or more bases are equally possible in thesequences shown in FIGS. 3A and 3B.

TABLE 1 Ambiguity codes for sugarcane polyubiquitin ubi4 gene sequencesupstream of the translation start codon Ambiguity Code Shown Position inFIG. 3A Actual Base 243 n G, A, T, or C 245 n G, A, T, or C 790 n G, A,T, or C 1023 n G, A, T, or C 1089 n G, A, T, or C 1174 n G, A, T, or C1656 n G, A, T, or C

TABLE 2 Ambiguity codes for sugarcane polyubiquitin ubi4 gene downstreamof the translation stop codon Ambiguity Code Shown Position in FIG. 3BActual Base 17 n G, A, T, or C 885 n G, A, T, or C 1055 s C or G 1059 yC or T 1463 n G, A, T, or C 1712 n G, A, T, or C 1769 r A or G 1810 w Aor T 1964 y C or T 2495 n G, A, T, or C

On subsequent sequencing of subclone pubi4, the nucleotide sequence (SEQID NO:5) and translated amino acid sequence (SEQ ID NO:6) weredetermined to be as shown in FIG. 5. The letter “X” in FIG. 5B refers toany amino acid. The organization of the ubi4 gene was determined asshown in FIG. 6.

SEQ ID NO:5 was cloned as two fragments into the plasmid vectorpBluescript II KS+(Stratagene) to generate plasmids pubi4a and pubi4b.Plasmids pubi4a and pubi4b were introduced into the host Escherichiacoli DH5alpha and the transformed Escherichia coli cells were depositedat the Agricultural Research Service Culture Collection (NRRL) under theterms of the Budapest Treaty on Mar. 8, 1999 as NRRLB-30112 (containingpubi4a) and NRRLB-30114 (containing pubi4b). NRRLB-30112 contains the2227 bp EcoRI/SalI fragment of SEQ ID NO:5, i.e., including the 1802 bp1802 bp SEQ ID NO:7, plus the first coding repeat and part of the secondrepeat. NRRLB-30114 contains the 3329 bp SalI/EcoRI fragment whichincludes the remainder of the coding region, the 3′ UTR, and furtherdownstream sequences.

An XbaI restriction site was added at the 3′ end of the ubi4 promotershown in SEQ ID NO:7 by way of PCR amplification with an XbaI adapterprimer. The polyubiquitin ubi4 promoter so modified was ligated upstreamof a GUS coding sequence and a NOS 3′ terminator in the vector plasmidpUC19 to form pubi4-GUS.

This plant expression plasmid was transformed into E. coli DH5a hostcells and deposited with the NRRL under the terms of the Budapest Treatyon Mar. 15, 1999 as NRRLB-30115.

Table 3 shows the ambiguity codes and the bases they represent in theubi4 sequences shown in FIGS. 5 and 10.

TABLE 3 Ambiguity codes for sugarcane polyubiquitin ubi4 and ubi9 genesAmbiguity Code Actual Base a A c C g G t/u T m A or C r A or G w A or Ts C or G y C or T k G or T v A or C or G h A or C or T d A or G or T b Cor G or T x/n G or A or T or C not G or A or T or C

Subclone pubi4 (FIGS. 3, 4, 5 and 6) contained four copies of thepolyubiquitin coding repeat, 238 bp of 3′ UTR, which is approximately95% identical to the corresponding region of the scubi241/511 cDNAs, apossible poly-A addition signal 215 bp 3′ of the TAA stop codon, andapproximately 2.6 kb of further downstream sequence. Immediately 5′ ofthe initiation codon was an intron of 1360 bp within SEQ ID NO:5 (intronof 1382 bp within SEQ ID NO:1) which was preceded by 65 bp in SEQ IDNO:5 (67 bp in SEQ ID NO:1) that were 98% identical to the 5′ UTRsequence of scubi241/511. The transcription start site has not yet beendetermined, and it is not known if the scubi241 and scubi511 cDNA clonescontain the entire 5′ UTR; however, since both scubi241 and scubiS 1cDNA clones started at the same nucleotide, this site can serve as aputative transcription start site for discussion purposes. The pubi4subclone contained an additional 377 bp upstream of the transcriptionstart codon, with a TATA consensus at −30 bp relative to the beginningof the cDNAs. Approximately 320 bp upstream of the transcription startcodon were two 10 bp sequences that showed homology to the heat stresspromoter element (HSE) consensus sequence (aGAAnnTTCt) [Scharf et al.:Heat stress promoters and transcription factors. In: Nover L (ed) PlantPromoters and Transcription Factors, pp. 125-162. Springer-Verlag,Berlin (1994)]. The second of these 10 bp sequences, however, lacked theG residue at position two that has been found to be invariant in HSEs[Scharf et al. (1994) supra].

Subclone pubi9 sequence and organization were initially determined to beas shown in FIGS. 7A, 7B and 4. Tables 4-5 provide the meaning ofsequence symbols other than the bases G, A, T, and C which indicatesambiguities where two or more bases are equally possible in thesequences shown in FIGS. 7A and 7B.

TABLE 4 Ambiguity codes for sugarcane polyubiquitin ubi9 gene upstreamof the translation codon Ambiguity Code Shown Position in FIG. 7A ActualBase 9 n G, A, T, or C 3613 n G, A, T, or C

TABLE 5 Ambiguity codes for sugarcane polyubiquitin ubi9 gene downstreamof the translation stop codon Ambiguity Code Shown Position in FIG. 7BActual Base 59 n G, A, T, or C 286 n G, A, T, or C 294 n G, A, T, or C319 n G, A, T, or C

On subsequent sequencing of subclone pubi9, the nucleotide sequence (SEQID NO:8) and translated amino acid sequence (SEQ ID NO:9) weredetermined to be as shown in FIG. 8. The letter “X” in FIG. 8B refers toany amino acid. The organization of the ubi9 gene was determined asshown in FIG. 6. Table 3, supra, shows the ambiguity codes and the basesthey represent in the ubi9 gene sequences shown in FIGS. 8 and 11.

An approximately 7.2 kb HindIII-EcoRI fragment, which contains SEQ IDNO:8 plus approximately 2kb additional downstream sequence, was clonedin the plasmid vector pBluescript II KS+(Stratagene) to form plasmidpubi9. This plasmid was transformed into E. coli DH5a host cells anddeposited with the Agriculture Research Service culture collection(NRRL), under the terms of the Budapest Treaty on March 8, 1999 asaccession number NRRLB-30113.

An XbaI restriction site was added at the 3′ end of the ubi9 promotershown in SEQ ID NO:10 by way of PCR amplification with an XbaI adapterprimer. The ubi9 promoter so modified was ligated upstream of a GUScoding sequence and a NOS 3′ terminator in the vector plasmid pUC19 toform pubi9-GUS. This plant expression plasmid was transformed into E.coli DH5a host cells and deposited with the NRRL under the terms of theBudapest Treaty on Mar. 15, 1999 as accession number NRRLB-30116.

Subclone pubi9 (FIGS. 4, 6, 7 and 8) contained five copies of thepolyubiquitin coding repeat, 244 bp of 3′ UTR, which were 98% identicalto the corresponding region of scubi241/511 and 95% identical to thecorresponding region of pubi4. A possible poly-A addition signal waspresent 221 bp down stream of the TAA stop codon, and there wasapproximately 2 kb additional downstream sequence. As with the ubi4gene, an intron was located immediately 5′ of the initiation codon; thisintron was 1374 bp. 5′ of this intron were 65 bp (in SEQ ID NO:8) and 67bp (in SEQ ID NO:3) that was 97% identical to both the 5′ UTR of thescubi241/511 cDNA clones and the corresponding region of the ubi4 gene.The subclone contained an additional 2247 bp of upstream sequence,including a TATA consensus sequence at−30 bp relative to the beginningof the cDNA clones. Upstream of the 5′ UTR, a 577 bp region of ubi9 fromposition 1671 to 2248 of SEQ ID NO:10 was highly homologous (>90%identity) to the corresponding region of ubi4 (positions 1 to 377 of SEQID NO:5). Partial sequence from an additional subclone of kubi4indicated that this high degree of homology continues at least as far as2kb upstream of the transcribed region of the genes (unpublished data).Within this highly homologous region, approximately 344 bp upstream oftranscription start codon, is an apparent insertion of approximately 200bp not present in the sugarcane ubi4 promoter. This 200 bp region wasdelimited by 17 bp imperfect inverted repeats. A 202 bp region 82%identical to this insertion is also found in the 3′ UTR of a sugarcaneglucose transporter cDNA clone SGT1 [Bugos et al., Plant Physiol. 103,1469-1470 (1993)]. The nature of this possible insertion event has notbeen investigated; however, it has features of miniature inverted-repeattransposable elements (MITEs) [Wessler et al., Curr Opin Genet Dev 5,814-21 (1995)]. Without limiting the invention to any particularmechanism, this insertion is not believed to have a functional role inthe promoter activity of the polyubiquitin ubi9 promoter since thisinsertion is inserted in the 3′ UTR (not the promoter) of the glucosetransporter gene, and since it is not present in the polyubiquitin ubi4gene. Like the ubi4 gene, the ubi9 gene also contained two HSE-likesequences about 320 bp upstream of the transcription start codon;however, both of these HSE-like sequences lacked the invariant Gresidue.

A comparison of the sequences upstream of the translation start codon ofthe sugarcane ubi4 gene sequence with the maize polyubiquitin promoter(GenBank accession number S94464; Quail et al., U.S. Pat. Nos. 5,614,399and 5,510,474) showed only 51% homology when comparing a fragmentcontaining the sequence upstream of the 5′ UTR, the 5′ UTR sequence andthe intron sequence, only 64% homology when comparing a fragmentcontaining the sequence upstream of the transcription start codon, only65% homology when comparing a fragment containing the 5′ UTR sequence,and only 58% homology when comparing a fragment containing the intronsequence.

A comparison of the sequences upstream of the translation start codon ofthe sugarcane ubi9 gene sequence with the maize polyubiquitin promoter(GenBank accession number S94464; Quail et al., U.S. Pat. Nos. 5,614,399and 5,510,474) showed only 61% homology when comparing a fragmentcontaining the sequence upstream of the 5′ UTR, the 5′ UTR sequence andthe intron sequence, only 63% homology when comparing a fragmentcontaining the sequence upstream of the transcription start codon, only66% homology when comparing a fragment containing the 5′ UTR sequence,and only 59% homology when comparing a fragment containing the intronsequence.

Some heat-shock-inducible polyubiquitin promoters have been found tocontain HSEs (Binet, et al., 1991, supra; Christensen et al. (1992)supra]. Both the ubi4 and ubi9 genes contained two short sequenceelements that have some homology to HSEs; however, three out of four ofthese elements lacked the G residue that has been found to be invariantat position 2 of the HSE (aGAAnnTTCt) (Scharf et al. (1994) supra].Given the very marginal induction (approximately 2-fold or less) of theubi4 and ubi9 genes when compared to other sugarcane polyubiquitingenes, it is doubtful that these HSE-like elements play a role inregulating gene expression. Similar observations have been made inconnection with the tobacco polyubiqutin promoter, Ubi.U4; while thispromoter also contains “. . . two degenerated heat shock-like elements.. , ” removal of these elements had no significant effect on geneexpression (Plesse, et al. (1997) supra].

Both the ubi4 and ubi9 genes contained a large intron immediatelyupstream of the protein coding region preceded by an approximately 65 bpfragment which was highly homologous to the 5′ UTR of scubi241/511. Anintron at this position (i.e., immediately upstream of the initiationcodon) has been found in many other plant polyubiquitin genes (Binet, etal., 1991, supra; Christensen et al. (1992) supra; Garbarino et al.(1995) supra; Norris et al., Plant Mol Biol 21, 895-906 (1993)], and insome cases been shown important for high levels of expression (Norris etal. (1993) supra; Garbarino et al. (1995) supra]. By analogy, it is theinventors' belief that the intron of the ubi4 and ubi9 genes may alsoplay a role in regulating gene expression.

The cDNA clones scubi241 and scubi511 contain three and five copies ofthe polyubiquitin coding sequence, respectively. Genomic clones pubi4and pubi9 contain four and five copies of the polyubiquitin codingsequence, respectively. Without limiting the invention to any particularmechanism, this may mean that the scubi241/511 sub-family contains atleast three different genes, containing three (i.e., scubi 241/511),four (i.e., pubi4) and five (i.e., pubi9) ubiquitin repeats.Alternatively, it is possible that the difference in the number ofcopies of the polyubiquitin coding sequence reflects a differencebetween the cultivars, with the scubi241/511 sub-family in H65-7052containing genes with three and five ubiquitin repeats, while the samesub-family in H32-8560 contains genes with four and five ubiquitinrepeats.

EXAMPLE 2 Construction of Plasmids pubi4-GUS, pubi9-GUS, 4PI-GUS and9PI-GUS Comprising Sugarcane Polyubiquitin Promoters And an ExemplaryStructural Gene

Reporter plasmids were made placing the uid A gene encodingβ-glucuronidase (GUS) [Jefferson et al., Proc. Natl. Acad. Sci. USA 83,8447-8451 (1986)] and the nopaline synthase (NOS) terminator under thecontrol of the sugarcane polyubiquitin promoter disclosed herein. Thepromoter sequence in plasmid pubi4-GUS contained nucleotides 1-1810 ofSEQ ID NO:1 (i.e., nucleotides 1-1802 of SEQ ID NO:5) and and XbaI site(TCTAGA) added immediately after bp 1802, by way of an adapter on a PCRprimer. The promoter sequence in plasmid pubi9-GUS contained nucleotides1-3688 of SEQ ID NO:3 (i.e., nucleotides 1-3688 of SEQ ID NO:8) and andXbaI site (TCTAGA) added immediately after bp 3688, by way of an adapteron a PCR primer. The Expands™ PCR system (Boehringer Mannheim) was usedto amplify part of the 5′ UTR and the intron including the 3′ splicesite; this PCR product was used to join the sequence upstream of the 5′UTR, the 5′ UTR, and intron to the GUS gene using a unique NruI site inthe 5′ UTR and an XbaI site added as an adapter to the 3′ PCR primer.

pHA9 contained the maize ubi1 promoter driving a neomycinphosphotransferase II (NPTII) gene and NOS terminator. It was made byremoving the luc gene from pAHC18 described in [Christensen et al.,Transgenic Research 5, 213-218 (1996)] as BamHI fragment and replacingit with an 844 bp BamHI fragment containing the NPII gene.

35S-GUS contained the uid A gene encoding GUS under the control of thecauliflower mosaic virus (CaMV) 35S RNA promoter sequence (Clontech).

Binary plasmids 9PI-GUS, 4PI-GUS and MPI-GUS for Agrobacteriumtransformation were made by ligating the promoter-intron-GUS-NOScassettes from pubi9-GUS, pubi4-GUS, and pAHC27 [Christensen et al.(1996) supra] respectively, as HindIII-EcoRI fragments, into theHindIII-EcoRI sites of pCAMBIA1300 [Roberts et al., RockefellerFoundation Meeting of the International Program on Rice Biotechnology,Malacca, Malaysia (1997)]. Binary plasmid pHW537 contained a putative 5′nuclear matrix attachment region (MAR) from λubi4, ubi9 promoter andintron, GUS, and 3′ terminator and putative 3′ MAR from λubi4 asHindIII-EcoRI fragment in the HindIII-EcoRI sites of pCAMBIA1300.

EXAMPLE 3

Transient Expression of Pubi4-GUS and Pubi9-GUS in Sugarcane SuspensionCultured Cells

Sugarcane suspension cell cultures (variety H50-7209) were maintained asdescribed by [Nickell et al., Physiol. Plant. 22, 117-125 (1969)]. DNAreporter plasmids (pubi4-GUS, pubi9-GUS,or pAHC27) were introduced intosugarcane suspension culture cells by particle bombardment as previouslydescribed [Klein et al., Nature 327, 70-73 (1987)] using a PDS1000Biolistic particle accelerator (BioRad) at 1100 psi. Controls were notbombarded with DNA. Transient assays were carried out in a randomizedcomplete block design with four treatments (promoters) and sixreplications. Each replication consisted of bombardment of five samples.Two days after bombardment, plant material was assayed for GUSexpression. Each sample was divided into two equal parts, one forhistochemical analysis and one for chemiluminescent measure of GUSenzyme activity using the GUS-Light kit (Tropix) and an MLX plate readerluminometer (DYNEX). GUS enzyme activity assays were performed accordingto the manufacturer's protocol, with 60 minutes incubation in GUSreaction buffer before chemiluminescence was measured. GUS activity wasexpressed as relative light units (RLU) per nanogram total protein[Bradford, Anal. Biochem. 72:248-254 (1976)]. From each experiment, thehighest and lowest values were discarded for each plasmid. Analysis ofvariance was performed on the data from each set of experiments; thoseexperiments which showed a significant effect (P≦0.05) from promotertreatments were further analyzed by a least significant difference test(P≦0.05) to identify which promoters produced significantly differentresults.

The results of histochernical staining and chemiluminescent GUS activityassays of sugarcane suspension culture cells which had been bombardedwith the reporter plasmids are shown in FIG. 9. FIG. 9A shows that theaverage number of blue foci detected after bombardment with GUSexpression plasmids containing the ubi9 promoter was higher thanobserved for either the ubi4 or maize polyubiquitin ubi1 [Christensen etal. (1996), supra] promoters. Because of high levels of variability, itcould not be determined from histochemical staining of these transientexpression experiments whether the results seen in sugarcane callus arein fact significantly different. However, using a chemiluminescent assayto measure GUS activity again indicated the average level of expressionwas higher for the ubi9 promoter than for the sugarcane ubi4 or maizeubi1 promoters (FIG. 9B). Statistical analysis indicated that thedifference, as measured by this assay, was significant at P≦0.05.

These data demonstrated that both the ubi4 and ubi9 promoters inpubi4-GUS and pubi9-GUS, respectively, were sufficient to directtransient expression in monocotyledonous sugarcane suspension cells.

EXAMPLE 4 Transient Expression of pubi4-GUS and pubi9-GUS in Tobaccoleaves

Tobacco cultivar Wisconsin 38 was grown in Magenta boxes on MSNT medium[1X Murashige and Skoog salts (GIBCO BRL #11117-074), 1X minimalorganics (GIBCO BRL #11118-023), 30g/l sucrose, 0.8% agar] at 26° C.under a 16 h light regime. DNA reporter plasmids (pubi4-GUS, pubi9-GUS,or pAHC27) were introduced into tobacco leaves by particle bombardmentas previously described [Klein et al., (1987) supra] using a PDS1000Biolistic particle accelerator (BioRad) at 650 psi. Controls were notbombarded with DNA. Transient assays were carried out in a randomizedcomplete block design with four treatments (promoters) and sixreplications. Each replication consisted of bombardment of five samples.Two days after bombardment, plant material was assayed for GUSexpression. Each sample was divided into two equal parts, one forhistochemical analysis and one for chemiluminescent measure of GUSenzyme activity as described supra (Example 3).

The results of histochemical staining and chemiluminescent GUS activityassays of tobacco leaves which had been bombarded with the reporterplasmids are shown in FIG. 9. The results show that average GUSexpression was higher for the sugarcane ubi4 promoter than for eitherthe maize polyubiquitin promoter or the sugarcane ubi9 promoter whenusing either the histochemical (FIG. 9C) or chemiluminescent (FIG. 9D)assays. Analysis of the chemiluminescent data indicates that thedifference between the sugarcane ubi4 and maize polyubiquitin promoterswas statistically significant at P<0.05.

These data demonstrated that both the ubi4 and ubi9 promoters inpubi4-GUS and pubi9-GUS, respectively, were sufficient to directtransient expression in dicotyledonous tobacco leaves.

EXAMPLE 5 Transient Expression of pubi4-GUS and pubi9-GUS in SorghumCallus

Sorghum immature embryo derived callus was cultured and bombarded aspreviously described with the reporter plasmid ubi4-GUS or ubi9-GUS(prepared as described in Example 2) and with several reporter plasmidsin which the uid A gene encoding GUS was placed under the control ofeach of several promoters including maize adh, 35S, 35S:35S, rice actin,and maize ubi1 as described below.

A. Culture media

N6 maintenance medium [Macro elements (mg/l final concentration), 2830KNO₃, 1650 (NH₄)SO₄, 166 CaCl₂-2H₂O, 185 MgSO₄-7H₂O, 400 KH₂PO₄; Microelements (mg/l final concentration), 37.3 Na₂ EDTA, 27.8 FeSO₄-7H₂O, 1.6H₃BO₃ 0.76 Kl, 3.3 MnSO4, 1.5 ZnSO₄-7H₂O; Carbohydrates (g/l finalconcentration), 20 Sucrose; Hormones (mg/l final concentration), 1.02,4-Dichlorophenoxyacetic acid; Vitamins (mg/l final concentration), 0.5Thiamine-HCl, 0.25 Pyridoxine-HCl, 0.25 Nicotinic Acid; Amino Acids(mg/l final concentration), 2875 L-Proline, 2.0 Glycine, 100 CasaminoAcids; and Agar (g/l final concentration), 2.5 Phytagel] was used.

B. Plant material

Highly embryogenic callus tissue derived from sorghum plants (Sorghumbicolor L. Moench, cv BWheatland 399) was used. To establish calluscultures, caryopses 10 to 18 d post-anthesis were surface-sterilizedwith 70% ethanol for 5 min and 20% clorox bleach for 15 min, followed bytwo changes of sterile distilled water. Immature embryos, 1.0 to 1.5 mmlong, were aseptically removed using a sterilized 11 cm forceps in alaminar flow hood under a stereo dissecting microscope. The embryos wereplaced with the scutella exposed on N6 medium modified for sorghum cellculture and solidified with 2.5 g/l Phytagel.

C. Microprojectile bombardment preparation

Prior to bombardment, 1 mm gold particles were coated with transformingDNA by the procedure of Daines (1990). A stock suspension of goldparticles was suspended at 60 mg/ml in absolute ethanol. Thirty-fivemicroliters of the suspension was transferred into a 1.5 mlmicrocentrifuge tube, centrifuged at 14,000 g for 3 min, and the pelletwas suspended in 200 μl of sterile distilled water. Following a secondcentrifugation, the pellet was suspended in 25 ml of Tris-EDTAcontaining 25 mg of the transforming plasmid DNA. The following chilledsterile solutions were added in order: 200 ml of water, 250 ml of 2.5 MCaCl₂, and 50 ml of 0.1 M Spermidine (0.2 μm filter-sterilized). Themicrocentrifuge tubes were shaken with a Tomy microtube shaker at 4° C.for 15 min and centrifuged at 16,000 g for 5 min. The supernatant wasremoved, the pellet washed with 200 ml of ethanol and the DNA-coatedgold particles suspended in 36 ml of ethanol.

D. Target tissue establishment and bombardment

Immature embryos were removed from sorghum caryopses and cultured on N6maintenance medium for 7 d. If the immature embryos are less than 0.5 mmthey may die in culture and if they are larger than 1.5 mm they mayprecociously germinate instead of initiating into callus tissue. Fourhours prior to bombardment, approximately 50 embryo-derived calli wereplaced in a circle (4 cm diameter) in the center of a Petri dish (15×100mm) containing 0.2 M mannitol and 0.2 M sorbitol in N6 maintenancemedium solidified with 2.5 g Phytagel. The Petri dish containing thetarget callus tissue was placed in the biolistic device and 10 ml of theDNA-gold suspension pipetted onto the center of a macroprojectile. Thedistance between the stopping plate and the target callus tissue wasadjusted to 13 cm. The tissue was bombarded under vacuum with therupture disk strength at 1100 p.s.i.

Callus tissue was sampled one day post bombardment using the GUShistochemical assay. Approximately twenty (20) replicates were performedfor each promoter. The average number of blue foci per bombarded platewas determined for each reporter plasmid using a stereo microscope.

The ubi4 nucleotide sequence was sufficient to direct transientexpression in monocotyledonous sorghum. Indeed, the ubi4 nucleotidesequences resulted in significantly higher levels of expression of GUSas compared to the levels of expression driven by each of the othertested promoters (data not shown). These results demonstrate that theubi4 promoter in pubi4-GUS was sufficient to direct transient expressionin monocotyledonous sorghum callus.

EXAMPLE 6

Transient Expression of pubi9-GUS in Pineapple Leaves, Protocorm-LikeBodies, Roots and Fruit

Pineapple cultivar F153 leaves, protocorm-like bodies (plbs), roots andfruit were bombarded with a reporter plasmid [pAHC27, pubi9-GUS or35S-GUS] described supra (Example 2). Target tissue (leaves, plbs, rootsand fruit) was plated in the center (2.5 cm diameter) of petri plates inmodified MS medium supplemented with 0.8% Difco Bacto agar and 3%sucrose. Bombardments were performed with a Bio-Rad helium gas-drivenmicroprojectile accelerator (PDS- 1000/He, Bio-Rad, Hercules, CA) with1100 psi rupture discs. Gold microcarriers (1.6-μm-diameter, Bio-Rad)were coated with DNA using the CaCl2 precipitation method following themanufacturer's directions. Two ug of each DNA construct were used foreach shot.

Histochemical GUS staining was performed 48 hours following bombardmentto determine transient transformion. Five (5) or three (3) replicateswere performed for each promoter in each type of bombarded tissue. Thenumber of blue foci/plate of each bombarded tissue is shown in Table 4.

TABLE 4 Number of Blue Foci Per Plate In F153 Pineapple Tissues PlasmidpAHC27 ubi9-GUS 35S-GUS Leaves 11.0 ± 7.0^(a) 244.0 ± 22.0 — PIbs 142.0± 45.2  150.3 ± 52.3 — Roots 3.2 ± 0.9  7.4 ± 2.2  1.6 ± 0.8 Fruit 8.7 ±6.4 11.0 ± 3.7 14.2 ± 8.1 ^(a)Standard error.

The above data demonstrate successful transient expression of GUS underthe control of the sugarcane polyubiquitin ubi9 promoter in pubi9-GUS ineach of the four tissues of monocotyledonous pineapple.

EXAMPLE 7 Stable Expression of pubi4-GUS and pubi9-GUS in TransgenicSugarcane Callus

Sugarcane callus cultures were initiated from stem apices (varietyH62-4671) by surface sterilizing the plant material with 70% ethanol,cutting two mm transverse slices and growing on MS2 plates [1× Murashigeand Skoog salts (GIBCO BRL #11117-074)], 1×minimal organics (GIBCOBRL#11118-023), 2 mg.L 2,4-D, 0.7% agar] under a 16 h light regime.After one to two months, callus was transferred to MS1 plates[1×Murashige and Skoog salts (GIBCO BRL #11117-074), 1×minimal organics(GIBCO BRL #11118-023), 1 mg/L 2,4-D, 0.7% agar] and subculturedmonthly.

Sugarcane callus was co-bombarded with a reporter plasmid (pubi4-GUS,pubi9-GUS, or pAHC27) and the selection plasmid pHA9 which contained themaize ubi1 promoter driving a neomycin phosphotransferase II (NPTII)gene (prepared as described supra in Example 2). Bombardment was withone micron gold particles and 1550 psi rupture discs. After bombardment,callus was kept on MS 1 plates without selection for two weeks. Afterhis recovery period, calli were transferred to MS 1 plates with 50 mg/LG418 (Agri-bio) for one month, then transferred to MS1 plates with 100mg/L G418 for 2-3 months. Calli which survived this selection weretransferred to MS1 plates with 60 mg/L G418 for multiplication. Smallcalli totaling about 50 were randomly chosen from selected lines. Thesecalli were assayed for GUS activity using the GUS-Light kit (Tropix)according tot the manufacturer's protocol, with 30 minutes incubation inGUS reaction buffer before chemiluminescence was measured. Two to ten 50mg samples were assayed from each line, with three chemiluminescenceassays for each sample.

The results of the GUS chemiluminescent activity assays are shown inFIG. 12. In ten selected independent transgenic sugarcane callus lines,GUS expression from the sugarcane ubi9 promoter averaged 535.4 RLU/ngprotein/30 min (FIGS. 12A, 12D). Seven selected stable transgenic linesexpressing GUS under the control of the sugarcane ubi4 promoter averaged209.3 RLU/ng protein/30 min (FIGS. 7B, & 7D), and seven lines expressionGUS under the control of the maize ubi 1 promoter averaged 348.2 RLU/ngprotein/30 min (FIGS. 7C, & 7D).

These results demonstrate that both the ubi4 and ubi9 nucleotidesequences in pubi4-GUS and pubi9-GUS, respectively, were sufficient todirect stable expression in monocotyledonous sugarcane callus.

EXAMPLE 8 Stable Expression of 9PI-GUS in Transgenic Rice Callus

Agrobacterium strain EHA105 [Hood et al. J. Bacteriol. 168:1291-1301(1986)] was used to transform rice callus. Reporter plasmids (9PI-GUS,4PI-GUS, MPI-GUS, or pHW537, which were prepared as described in Example2, supra] were introduced into Agrobacterium strain EHA105 by a standardprocedure.

Rice callus was induced from scutellum tissue of rice (cv. Taipei 309)and transformed by Agrobacterium co-cultivation as previously described[Hiei et al., (1994) supra]. After 2-3 months on selection mediumcontaining 100 mg/l hygromycin B (CalBiochem), small calli from selectedlines were assayed for GUS activity by the chemiluminescence methoddescribed supra (Example 3).

PCR using one primer within the T-DNA and one outside of the T-DNA rightborder was used to confirm the absence of Agrobacterium contamination intested callus lines using methods known in the art. Because ricetransformation was by Agrobacterium, there is the possibility that theAgrobacterium were not killed after transformation, and thus that theGUS expression seen is from the Agrobacterium, not from transgenic ricecells. To test for this possibility, two PCRs were performed: one totest for the presence of the GUS gene, the second to test for thepresence of vector plasmid sequences outside of the T-DNA. TheAgrobacterium harbor a binary plasmid, one section of which contains theGUS encoding gene under the control of sugarcane promoter sequences.This section of the vector plasmid is called the T-DNA, and only thispart is ordinarily transferred to the plant genome. A positive PCR forGUS indicates that the isolate plant DNA may be successfully amplifiedby PCR and the GUS gene is pressent (i.e., confirming the observation ofGUS activity). A negative PCR for the vector plasmid outside of theT-DNA confirms that the entire plasmid, which is what is present in theAgrobacterium, is no longer present, i.e., that the T-DNA (whichcontains the GUS sequences under the control of the sugarcane promotersequences) is successfully integrated into the plant genome.

One, seven, four, and two stably transformed transgenic rice calluslines were selected following co-cultivation of rice callus withAgrobacterium which had been transformed with the 4PI-GUS, 9PI-GUS,pHW537, and MPI-GUS reporter plasmids, respectively. GUS expression insix transgenic lines transformed with the ubi9 promoter sequence isshown in FIG. 13. GUS expression in six transgenic lines transformedwith the ubi9 promoter sequence averaged 681.0 RLU/ng protein/30 min.

Four stably transformed transgenic rice callus lines were selectedfollowing co-cultivation of rice callus with Agrobacterium which hadbeen transformed with the pHW537 reporter plasmid. GUS expression inthese transgenic lines averaged 567.7 RLU/ng protein/30 min. Since thepHW537 reporter plasmid differed from the 9PI-GUS plasmid inadditionally containing the putative 5′ and 3′ flanking nuclear matrixattachment regions (MARs), and since both the 9PI-GUS and pHW537reporter plasmids successfully resulted in expression of comparablelevels of GUS in rice callus, these results demonstrate that theputative 5′ and 3′ flanking nuclear MARs are not necessary for promoteractivity of the pubi9 sequences.

The results also suggest that either the polyubiquitin ubi4 or ubi9promoter (with or without putative MARs) drives GUS expression at levelscomparable to the maize ubi1 promoter in stable transgenic rice callus.

The above results demonstrate that the ubi9 promoter in 9PI-GUS, andpHW537 was sufficient to direct stable expression in monocotyledonousrice callus.

EXAMPLE 9 Stable Expression of pubi4-GUS and pubi9-GUS in TransgenicTobacco leaves

Agrobacterium tumefaciens was used to transform tobacco leaves. Reporterplasmids (9PI-GUS, 4PI-GUS, MPI-GUS, or 35S-GUS, which were prepared asdescribed in Example 2, supra), were introduced into leaf discs by anAgrobacterium mediated transformation procedure adapted from Horsch etal Science 227:1229-1231 (1985). Controls were untransformed.

Briefly, two ml overnight cultures of Agrobacterium tumefaciens werepelleted by brief centrifugation, decanted and resuspended in two ml ofMSNTS liquid media (per liter of medium: one package MS salt mix [GIBCOBRL #11117-066], 30 g sucrose, 1.0 ml 1000×B5 vitamins, 50 μg/mlα-naphthaleneacetic acetic acid [GIBCO BRL #21570-015], and 50 μl 20mg/l benzyladenine [GIBCO BRL #16105-017]).

Aseptically grown tobacco leaves were harvested and placed in petriplates containing 20 ml MSNTS with 0.6 ml of the resuspendedAgrobacterium and cut into approx. 1 cm squares with a sterile scalpel.After one to five minutes the leaf pieces were removed from the liquid,gently blotted on dry sterile paper, and placed on 0.8% agar MSNT plates(per liter of medium: one package MS salt mix [GIBCO BRL #11117-066], 30g sucrose, 1.0 ml 1000×B5 vitamins, 8 g Bacto-Agar) with 500 μg/mlCefotaxime (PhytoTechnology Laboratories). After two days, the leafpieces were transferred to 0.8% agar MSNTS plates with 500 μg/mlCefotaxime and 100 μg/ml Kanamycin (PhytoTechnology Laboratories).Shoots appeared after two to three weeks, at which time the shoots weretransferred to Magenta boxes containing MSNT media with no antibiotics.To confirm Kanamycin resistance and to reduce the likelihood ofobtaining chimeric plants, a “shooting assay” was used: leaves fromputative transgenic plants were placed on 0.8% agar MSNTS platescontaining 100 μg/ml Kanamycin. Shoots arising from these “shootingassays” were transferred to non-selective rooting media (MSNT) and grownin Magenta boxes.

Expression of GUS was determined as described in Example 3, supra. Theresults demonstrate that the both the ubi4 and ubi9 nucleotide sequencesin 4PI-GUS and 9PI-GUS, respectively, were sufficient to direct stableexpression in dicotyledonous tobacco leaves. Furthermore, The levels ofGUS expression under the control of either the ubi4 and ubi9 nucleotidesequences in 4PI-GUS and 9PI-GUS were equal to or greater than thelevels of GUS expression under the control of the maize ubi1 promoter.On the other hand, expression of GUS under the control of the CaMV 35 Spromoter was greater than that under the control of either the sugarcaneubi4 or ubi9 promoters (data not shown).

EXAMPLE 10 Stable Expression of pubi4-GUS and pubi9-GUS in TransgenicMaize Embryos and Regeneration of Transgenic Maize Plants

Embryogenic corn cultures are initiated from immature maize embryos,bombarded simultaneously with a reporter plasmid (pubi4-GUS, pubi9-GUS,or mzubi1-GUS) and a selection plasmid (pHA9), and stably transformedcorn plants regenerated as previously described by Brown et al., U.S.Pat. Number 5,593,874, incorporated by reference.

Briefly, embryogenic corn cultures are initiated from immature maizeembryos of the “Hi-Ir” genotype which had been cultured 18-33 days on N62-100-25 -Ag medium modified to contain 2 mg/L 2,4-dichlorophenoxyaceticacid, 180 mg/L casein hydrolysate, 25 mm L-proline, 10 μM silvernitrate, pH5.8, solidified with 0.2% Phytagel™ (Sigma). Theseembryogenic cultures are used as target tissue for transformation byparticle gun bombardment.

A 1:1 mixture of the reporter vector (pubi4-GUS, pubi-GUS or mzubi1-GUS) and selection plasmid (pHA9) is precipitated onto tungsten M10particles by adding 12.5 μl of particles (25 mg/ml in 50% glycerol), 2.5μl plasmid DNA (1 μg/μl), 12.5 μ1M calcium chloride, and 5 μl 0.1Mspermidine, and vortexing briefly. The particles are allowed to settlefor 20 minutes, after which 12.5 μl of supernatant is removed anddiscarded. Each sample of DNA-tungsten is sonicated briefly and 2.5 μlis bombarded into the embryogenic cultures using a PDS-1000 Biolisiticsparticle gun (DuPont).

The bombarded tissue is transferred to fresh, nonselective medium theday after bombardment. Six days post-bombardment, the material istransferred to selective media containing 50 mg/L G418 (Agri-bio). After2-3 weeks, the cultures are transferred to fresh media which contains200 mg/L G418. The cultures are maintained on the 200 mg/L G418 media,transferred at 2-3 week intervals, until G418-resistant calli could bedistinguished. G418-resistant calli are recovered from the embryogenicmaterial. G418-resistant lines are bulked up and assayed for GUSexpression using a histochemical or chemiluminescent assay as describedsupra (Example 3).

Plants are regenerated from the G418-resistant calli which express GUSactivity in a three step regeneration protocol. All regeneration isperformed on 200 mg/L G418. The first two steps are carded out in thedark at 28° C., and the final step under a 16:8 hour photoperiod, atabout 25° C. Small green shoots that formed on Regeneration Medium 3 in100×25 mm Petri plates are transferred to Regeneration Medium 3 in200×25 mm Pyrex™ or Phytatrays™ to permit further plantlet developmentand root formation. Upon formation of a sufficient root system, theplants are carefully removed from the medium, the root system washedunder running water, and the plants placed into 2.5″ pots containingMetromix 350 growing medium. The plants are maintained for several daysin a high humidity environment, and then the humidity is graduallyreduced to harden off the plants. The plants are transplanted from the2.5″ pots to 6″ pots and finally to 10″ pots during growth.

Corn plants regenerated from 418-resistant embryogenic calli whichexpress GUS activity are tested for GUS expression using histochemicalof chemiluminescent assays as described supra (Example 3). GUSexpression (e.g., as determined by blue staining in the histochemicalassay or by luminescence in the chemiluminescent assay) by one or moretissues of corn plants which are generated from calli that had beenbombarded with pubi4-GUS or pubi9-GUS demonstrates that the ubi4 andubi9 nucleotide sequences in pubi4-GUS and pubi9-GUS, respectively, aresufficient to direct stable expression in regenerated monocotyledonousmaize plants.

EXAMPLE 11 Stable Expression of 4PI-GUS and 9PI-GUS in Transgenic TomatoPlants

A reporter plasmid (4PI-GUS or 9PI-GUS, prepared as described in Example2) or a control plasmid (i.e., a plasmid which contains the uid A geneencoding GUS and which lacks a promoter sequence) are transformed intoAgrobacterium, and the transformed Agrobacterium is used to infecttomato plants as previously described in Theologis et al., U.S. Pat. No.5,723,766, incorporated by reference.

Briefly, a reporter plasmid or control plasmid is introduced intoAgrobacterium strain LBA4404 as follows: Agrobacterium tumefaciensLBA-4404 (2 ml) is grown overnight at 28° C. in LB broth, and this usedto inoculate 50 ml of LB broth to obtain the desired culture. Theinoculated medium is grown at 28° C. until the OD.₆₀₀ is 0.5-1.0. Thecells are collected by centrifugation and the pellet is resuspended in 1ml, 20 mM ice cold CaCl₂. To 100 μl of the cell suspension, 1 μg of theplasmid is added, and the mixture is incubated on ice for 30 min beforesnap-freezing in liquid nitrogen. The cells are then thawed at 37° C.for 5 min and used to inoculate 1 ml LB. After 2 h growth at 28° C. withagitation, 100 μl of the culture are plated on LB+Kanamycin₅₀ medium;colonies are expected to appear in 2-3 days at 28° C. The cells arerecultured by picking several colonies and streaking on LB+Kanamycin₅₀medium; again, 3-4 colonies are picked from independent streaks and 5 mlcultures in LB+Kanamycin₅₀ medium are grown. Stationary phase culturesare used for transformion of tomato plants which are grown as describedinfra. Transformed Agrobacterium cells are frozen using 15% glycerol at−80° C. for later use.

To prepare host tomato plants, tomato seeds are sterilized using aprotocol which consists of treatment with 70% ethanol for 2 min withmixing; followed by treatment with 10% sodium hypochlorite and 0.1% SDSfor 10 min with mixing, followed by treatment with 1% sodiumhypochlorite, 0.1% SDS for 30 min with mixing, and washing with sterilewater 3 times for 2 min per wash. For germination of the sterilizedseeds, 0.8 g of the sterilized seeds are placed in a Seed GerminationMedium and grown for 2 weeks at low light in a growth room. After twoweeks, when the seeds had germinated, cotyledons are dissected from theseedlings by cutting off the cotyledon tips and then cutting off thestem. This process is conducted in a large petri dish containing 5-10 mlof MSO medium.

Feeder plates are prepared from a tobacco cell suspension in liquidmedium at 25° C. prepared with shaking at 130-150 rpm. The suspension istransferred to fresh medium at 1:10 dilution every 3-5 days. 1 ml ofrapidly dividing culture is placed on the feeder plate, overlaid withfilter paper and placed in low light in a growth room. The feeder platesare supplemented with 10 ml Feeder Medium Transformed Agrobacteriumcultures which contain the reporter or control plasmids are inoculatedinto 50 ml LB containing kanamycin with a single colony of the strain.The culture is grown by shaking vigorously at 30° C. to saturation(OD>2.0 at 600 nm). The strain is chosen to come to full growth in lessthan 24 h. The culture is then diluted 5 times and split into 50 mlportions in plastic tubes.

Cotyledons from two of the feeder plates are scraped into each tube androcked gently for 10-30 min. The cotyledons are then removed from thebacterial culture onto sterile filter paper (abaxial side up) on atobacco feeder plate and incubated for 48 h in low light in a growthroom. The cotyledons are then transferred axial side up to callusinducing medium. In the Callus inducing Medium, approximately fourplates are used per magenta box, and the explants are crowded. The boxis placed in a growth room for three weeks, and small masses of callusare expected to form at the surface of the cotyledons. The explants aretransferred to fresh plates containing the callus inducing medium everythree weeks. When the calli exceed 2 ml, they are transferred to platescontaining shoot inducing medium. When the stem structure is evident,the shoots are dissected from the calli and the shoots are transferredto plates containing root inducing medium. After a vigorous root systemis formed on the plants, the plantlets are transferred to soil by takingthe plantlets from the plates, removing as much agar as possible andplacing the plantlets in a high peat content soil in a small peat potwhich fits into a magenta box with cover. When the seedling leaves reachthe top of the box, the lid is loosened to uncover the box slowly over aperiod of 4-5 days. The plants are then transferred to a light cart andlarger pots, and kept moist. Flowers of these regenerated plants arepollinated and tomatoes are developed.

Expression of GUS is measured in regenerated tomato plant tissuestransformed with the reporter or control plasmids using a histochemicalor chemiluminescent assays as described supra (Example 3). GUSexpression by one or more tissues of tomato plants which are generatedfrom tissue that had been transformed with 4PI-GUS or 9PI-GUSdemonstrates that the ubi4 and ubi9 nucleotide sequences in 4PI-GUS and9PI-GUS, respectively, are sufficient to direct stable expression inregenerated dicotyledonous tomato plants.

EXAMPLE 12 Stable Expression of pubi4-GUS and pubi9-GUS in TransgenicSoybean Excised Embryonic Meristems And Regeneration Of TransgenicSoybean Plants

Soybean explants are derived from excised meristems, bombardedsimultaneously with a reporter plasmid (pubi4-GUS, pubi9-GUS, or mzubi1-GUS) and a selection plasmid (pHA9), and stably transformed soybeanplants are regenerated as previously described by Christou et al., U.S.Pat. No. 5,015,580, incorporated by reference.

Briefly, soybean explants of cultivar Williams 82 are derived frommeristems excised from the embryonic axes of immature seeds. Primaryleaves are removed and the explant plated on a target plate containing1% water agar. The explants are transformed with a reporter plasmid or aselection plasmid loaded at 1.0-0.001 μg/ml of beads. The particleaccelerator is charged at 13-16 kV. The carrier is loaded with 0.05-0.40mg of loaded beads per square centimeter, with a preferred level ofloading of 0.2 mg/cm².

The bombarded explants are then plated in the dark on modified MS basalmedium which has a high level (i.e., 13.3 μM) of the cytokininbenzylaminopurine. Following incubation of 1 to 2 weeks in the dark, thetissues are transferred onto the same basal medium at a lower (1.7 μM)level of cytokinin to promote shoot elongation. Shoots are harvested at0.5 to 1 cm in height.

The success of the transformation protocol is verified by fixingtransformed explants at each stage to assay for GUS activity asdescribed supra (Example 3). Two days after DNA particle injection,dozens of GUS active cells are expected to be detected in each explant.At 6 to 8 weeks, the plants are assayed for GUS activity in the shoot.Most plants are expected to be chimeric, having streaks of blue (i.e.,GUS-expression cells) when assayed using the histochemical assay.

The transformed excised meristem tissue is used to regenerate fullymature, and sexually mature chimeric plants using methods known in theart such as those described in U.S. Pat. No. 5,015,580 to Christou etal. (incorporated by reference). GUS expression by one or more tissuesof soybean plants which are regenerated from excised embryonic meristemsthat had been transformed with pubi4-GUS or pubi9-GUS demonstrates thatthe ubi4 and ubi9 nucleotide sequences in pubi4-GUS and pubi9-GUS,respectively, are sufficient to direct stable expression in regenerateddicotyledonous soybean plants.

From the above, it is clear that the invention provides promotersequences which are capable of driving transgene expression in bothmonocotyledonous and dicotyledonous plant cells, and which are usefulfor generating transgenic plants with desirable agronomiccharacteristics.

12 1 1813 DNA Saccharum Hybrid Cultivar H32-8560 misc_feature (243) The“n” at position 243 is any nucleotide. 1 gaattcatta tgtggtctaggtaggttcta tatataagaa aacttgaaat gttctaaaaa 60 aaaattcaag cccatgcatgattgaagcaa acggtatagc aacggtgtta acctgatcta 120 gtgatctctt gcaatccttaacggccacct accgcaggta gcaaacggcg tccccctcct 180 cgatatctcc gcggcgacctctggcttttt ccgcggaatt gcgcggtggg gacggattcc 240 acnanaccgc gacgcaaccgcctctcgccg ctgggcccca caccgctcgg tgccgtagcc 300 tcacgggact ctttctccctcctcccccgt tataaattgg cttcatcccc tccttgcctc 360 atccatccaa atcccagtccccaatcccat cccttcgtcg gagaaattca tcgaagcgaa 420 gcgaatcctc gcgatcctctcaaggtactg cgagttttcg atccccctct cgacccctcg 480 tatgtttgtg tttgtcgtacgtttgattag gtatgctttc cctgtttgtg ttcgtcgtag 540 cgtttgatta ggtatgctttccctgttcgt gttcatcgta gtgtttgatt aggtcgtgtg 600 aggcgatggc ctgctcgcgtccttcgatct gtagtcgatt tgcgggtcgt ggtgtagatc 660 tgcgggctgt gatgaagttatttggtgtga tctgctcgcc tgattctgcg ggttggctcg 720 agtagatatg gatggttggaccggttggtt cgtttaccgc gctagggttg ggctgggatg 780 atgttgcatn gcgccgttgcgcgtgatccc gcagcaggac ttgcgtttga ttgccagatc 840 tcgttacgat tatgtgatttggtttggact tattagatct gtagcttctg cttatgttgc 900 cagatgcgcc tactgctccatatgcctgat gataatccat aaatggcagt ggaaatcaac 960 tagttgattg cggagtcatgtatcagctac aggtgtaggg actagctaca ggtgtaggga 1020 ctngcgtcta attgtttggtccttaactca tgtgcaatta tgcaatttag tttagatgtt 1080 tgttccaant catctaggctgtaaaaggga cactggttag attgctgttt aatcttttta 1140 gtagattata ttatattggtaacttattaa cccntattaa catgccataa cgtggattct 1200 gctcatgcct gatgataatcatagatcact gtggaattaa ttagttgatt gttgaatcat 1260 gtttcatgta cataccacggcacaattgct tagttcctta acaaatgcaa attttactga 1320 tccatgtatg atttgcgtggttctctaatg tgaaatacta tagctacttg ttagtaagaa 1380 tcaggttcgt atgcttaatgctgtatgtgc cttctgctca tgcctgatga taatcatata 1440 tcactggaat taattagttgatcgtttaat catatatcaa gtacatacca tggcacaatt 1500 tttagtcact taacccatgcagattgaact ggtccctgca tgttttgcta aattgttcta 1560 ttctgattag accatatatcaggtattttt ttttggtaat ggttctctta ttttaaatgc 1620 tatatagttc tggtacttgttagaaagatc tggttncata gtttagttgc ctatccttcg 1680 aattaggatg ctgagcagctgatcctatag ctttgtttca tgtatcaatt cttttgtgtt 1740 caacagtcag tttttgttagattcattgta acttatgttc gcttactctt ctggtcctca 1800 atgcttgcag atg 1813 22804 DNA Saccharum Hybrid Cultivar H32-8560 misc_feature (17) The “n” atposition 17 is any nucleotide. 2 taatcctggg ccatgancag ctgtccttccaggttcacaa gtctggtgcc ttcttctgtc 60 cctccgatgg agattatctg catgtcgtggtcgtgtcctg atcgaatcct cgttgaatcc 120 ctatgttttt cttcaagaaa tgtgagtcctatgtcagtct ggttgcgttt gtgaacattt 180 ctgctgctga gcagcacttt ggctggaactgtgcaatgaa ataaatggaa ccctggtttc 240 tggttatgtg tgtgttagct aatgtttttgaagtggaagc tctaatcttc tatcgcgttg 300 ctactacaat tctgcttgtg ttttgatggttcttggtttc tgttagttgg ttcagaggaa 360 gttttgcttc cacagactaa gatgcagttgaactttggtt gccctggttt ctagatttca 420 tttgtgctgg ttgagtgata gtaagaaacaaccggtgttc acatataatc aggttttgtg 480 ctgctcgagt gatcgtcaaa aaccaccggtgttcacatct aaaaaggttt cgatccccag 540 gtttagatct cccgtttaat tccaaaaaaaaagttctgtg tacttgcatt tagttgggtg 600 gttgatgctg gaaagagtaa ctttcaagagtaataatctt tggtgactac tctgtttcaa 660 ctgatcaatc cctaggaaag gtacacctttacttagggaa gaaattctta gaaccttgca 720 ctttgtttca actgataata gtatactttattagataaaa aatattcaga tatattagac 780 accggatgtc atccactcat ccttacaaacctctgtcatg gtcctgcaga aatgtttgcc 840 agctccagtg gcttcctgat aaatctgtggagtgcctgtt aatcngctgc caatttttgc 900 tgagcactgt atatatgtta gtaagtactattgggccacc aattccattt tgacacagca 960 ctattggtcc accaattcga tcctgacacagcactgcata atttgaaacg tttttgctcc 1020 cattttgcaa ggctacaaat ttagatcatgtttascatyc tgtgggatac aatatatgga 1080 tatcgaacaa acttggtatg tcagagaaaaaatagtttat tttcaaaact aacattttta 1140 aagccttcta tgaactttaa accttcagcatttgggatca agatgagtgc tcgaacaaga 1200 gtgcactttt tctccaaaat aatctactacagagttcttt tttatatata aaaaaactta 1260 tacttaacag ataaatcaga cctcttctgctccatatcac cttgacaaat caaagaagca 1320 gcaccagcga agggtattat tattgaggtaaatataagat ctcgtttact gaaaaagacc 1380 gcgtgtttac ctaaactacc attttgctttgatagcagca tacatgtgat agaattgcgg 1440 atcctaccgt gctgactgtg aangtggtaagggtgagaga ttggtgggcg aggtctgaac 1500 gagcgaaaac agtactgcat ttactgttcacaaggaggcg gcttaggttt tggtctccca 1560 gctctctaag ggaagctgag aattatgattctcttgctta attatttctt aaccaaagtt 1620 ataaatatat agcctatgag atcctaatttatggaaataa ctaaactatt ttaaggaaat 1680 atataaatag ataatcagcc cactaacgggcntagcgccc actaacaggc ctggtgctga 1740 gcccgacata acatctctcc ccgcctggrgaaacagctcg tcctcgagct gaaatctggt 1800 agaagcatcw tcaaccaaca ccggggtcatgctggaacac tgcatcaggc gctaccgcag 1860 ctggtacgtc gtcgtcgagg aagtcagccgactccaagta gaacagtcgc ttacaactga 1920 tgtccgcgga cgtagggctc atcacaattgtaaacaaagc cctygacgac ggcactccaa 1980 acagctcttc cggtgtgaga cgacgaaacgagggagctag agcgggtagt ggcgcgggga 2040 acagccagtg tagcgcctgt agtcaccgagggatggggcg gtcgggcgcc gcgaggctgc 2100 ggtgccaggt ggaggttcaa cattcttcaaacgcccgtgc caagtacatg gcggactgga 2160 ggtcgggcgg tgcgcggagc tgaacctgcttacggaggtg gtccggcagc ccacccacgt 2220 acaactccgc cttttggcga gcggagaggttgtgggcatg gcacaggacg gcgttgtaac 2280 gctccgagta atcctgaacg gaagaaccaaaaggaaggcg ggcaagctcc gccaaccgag 2340 tgcccaaaac aggaggcccg aagcgaagcgagcataattc gcggaagcgc tcccaaggag 2400 gcataccctc gtcttgctcc agggcgtagtaccatgtctg ggcaacaccc cgaagatggt 2460 aggacgcgag ccatgtgcga gcggaggcgagcgtntgctg gccgcggaag aactgctcgc 2520 actggttcaa ccaattcagg ggatcggtcgaaccgtcgta cgtagggaac tccagtttgt 2580 agaatttggg ccccgcctgg gcgcctgcgagggcagcagc aagggctggg tcgagccccc 2640 cctgcggctg gccgcccgag ggagagggcgcccgaagaac agcggcccgt ccaccccccc 2700 gaaaagagtg ctggcggggg gtagggaggacatcgttgtc gccgccgccg tcgtgtaggc 2760 tgtcggggag ggcgacgtgg ccatcgagtagatccgggga attc 2804 3 3691 DNA Saccharum Hybrid Cultivar H32-8560misc_feature (9) The “n” at position 9 is any nucleotide. 3 aagttttgntaaaatgaaca aagaattggg gaaactatag ccaaagtggg tggggaatgg 60 tgccaaacaaaacttcgtaa accaacccaa aaagatccgg aaaacaaatg gatacgtgca 120 gggcatgcatgcaatagccc agccataaaa agcggcgagc caatgcccgg gtgtcaaaca 180 aaatggcgcctgtgccggct ctggctgctt ccggctcagc tttcggaacg atccgccgca 240 gtttggcctcgcatatgatg acgatgatgg tctcctcttc tcgatttgta gctccggcat 300 gggagccacctcctgtcggc tcacacatag cacgcgcctt agcccgtgct cgctctcccc 360 tagatgcttcacctgcgcca atcagtgtga gcccatcgtg tcagatggta ctcgtacgta 420 tggagtaacgtgataccaca acacgtacac tggtcagaat tgatagtata tgatcctgtc 480 gacccgatgtgttttagtac cttgcagtgg ccggagagga gtggccgcgc gcatgcggcg 540 caggggttctccgcgctcgc tgatcgcttc ctcactgtgc gctcgtttag gaacaccacc 600 tcgtggtcgctcaccatgtg tgactgcatg caacgctacg aatcaggacc cagatggaaa 660 cgaagcgcctctcgaccacc tctgcctcgg tgatggttgg tgtgcagtgc gtacgcatgc 720 acgctaccaatatcatacct ggatgccggt gcaatcgaac agcttcaggt tgtcgacgcg 780 gacggcgaagcaggacgcgt acttccatat ctttgggttc cattacgtac cgtcaatcga 840 ataaataaagagaagagttt gagatcagct tgttgggagc aggtgaccgc ccgacatgca 900 tgccgattgtcgacggcacg gaaataaaca acacatttgt gagggagcca gggaggcagt 960 ggcggcacagcgtcgcggca cagtcgatgc agaagtggtt cttgtcgttc ttgcgctccc 1020 cccgggtgtgcagcgcacgc ctttgaaaaa ctccgatagc aggccacaca gccattgcgg 1080 ggcgccgcgcacggccgcca gctgcatccc cgtttgttcg cacatgcgct aggtggtcct 1140 gcggccgttccttgcaccgc ggagacgcgg ggtggaccag tgggggaatg gatgaactgc 1200 tggtaggtttggttggattg gcgagtgcgt agagggggca tgggcaacga tagactcgat 1260 tcaattcaaagactgaaaat agtggagttc taacaccatt ctgtgcggcg ctaattctcg 1320 acatggcaggcgtaagcata ataccgacat ggcatgcaac gatgttcgtg aacagtggtg 1380 acacatggatatggtggccg tccaggggat tcgttccatt caattcaaag accgaaaatc 1440 gcggggttccgtagcatttt gtgcggtgct aattctcgaa catgcgagac gtaagcctaa 1500 taccgagatggcatgcaaca atgttcgtga acaacagtga cacgtggatg cggtggccgt 1560 ctagggattcgcgttctaag ctggtatatg tgcggtgtta attcttgaca tgcggggcgt 1620 aagtgtaataccaagatgaa cggtgacacg tggacgcggg ggtcgtcaaa caattcattc 1680 cgtggtctagggtaggttat atataaaggc cagtcttagt gggggatttt atggccatgt 1740 tattaatgcaacccatattt ggaaaacagt gcaggaagag tttcatcttc gtaaaactct 1800 ctctaattccatgaaactct tatcatctct ctcttcatca atacggtgcc acatcagcct 1860 atttaatgtccatgaaactc tgatgaaatc cactgagacg ggcctcagaa aacttgaaat 1920 cttctaaaaaaaattcaagt ccatgcatga ttgaagcaaa cggtatagca acggtgttaa 1980 cctgatctagtgatctcttg taatccttaa cggccaccta ccacaggtag caaacggcgt 2040 ccccctcctcgatatctccg cggcggcctc tggctttttc cgcggaattg cgcggtgggg 2100 acggattcctcgagaccgcg acacaaccgc ctttcgccgc tgggccccac accgctcggt 2160 gccgtagcctcacgggactc tttctccctc ctcccccgct ataaattggc ttcatcccct 2220 ccttgcctcatccatccaaa tcccagtccc caatcccagc ccatcgtcgg agaaattcat 2280 agaagcgaagcgaatcctcg cgatcctctc aaggtagtgc gagttttcga ttcccctctc 2340 gacccctcgtatgctttccc tgtttgtgtt tcgtcgtagc gtttgattag gtatgctttc 2400 cctgtttgtgttcgtcgtag cgtttgattt ggtatgcttt ccccgttcgt gttcctcgta 2460 gtgtttgattaggtcgtgtg aggcgatggc ctgctcgcat ccttcgatct gtagtcgatt 2520 tgcgggtcgtggtgtagatc tgcgggctgt gatgaagtta tttggtgtga tcgtgctcgc 2580 ctgattctgcgggttggctc gagtagatat gatggttgga ccggttggtt tgtttaccgc 2640 gctagggttgggctgggatg atgttgcatg cgccgttgcg cgtgatcccg cagcaggact 2700 tgcgtttgattgccagatct cgttacgatt atgtgatttg gtttggactt tttagatctg 2760 tagcttctgcttatgtgcca gatgcgccta ctgctcatat gcctgatgat aatcataaat 2820 ggctgtggaactaactagtt gattgcggag tcatgtatca gctacaggtg tagggactag 2880 ctacaggtgtagggacttgc gtctaaattg tttggtcctg tactcatgtt gcaattatgc 2940 aatttagtttagattgtttg ttccactcat ctaggctgta aaagggacac tgcttagatt 3000 gctgtttaatctttttagta gattatatat tatattggta acttattacc cttattacat 3060 gccatacgtgacttctgctc atgcctgatg ataatcatag atcactgtgg aattaattag 3120 ttgattgttgaatcatgttt catgtacata ccacggcaca attgcttagt tccttaacaa 3180 atgcaaattttactgatcca tgtatgattt gcgtggttct ctaatgtgaa atactatagc 3240 tacttgttagtaagaatcag gttcgtatgc ttaatgctgt atgtgccttc tgctcatgcc 3300 tgatgataatcatatatcac tggaattaat tagttgatcg tttaatcata tatcaagtac 3360 ataccatggcacaattttta gtcacttaac ccatgcagat tgaactggtc cctgcatgtt 3420 ttgctaaattgttctatttc tgattagacc atatatcatg taattttttt tttgggtaat 3480 ggttctcctattttaaatgc tatatagttc tggtacttgt tagaaaaatc tgcttccata 3540 gtttagttgcttatccctcg aattatgatg ctgagcagct gatcctatag ctttgtttca 3600 ggtatcaattctngtgttca acagtcagtt tttgttagat tcattgtaac ttatggtcgc 3660 ttactcttctggtcctcaat gcttgcagat g 3691 4 343 DNA Saccharum Hybrid CultivarH32-8560 misc_feature (59) The “n” at this position is any nucleotide. 4taagtcctgg gccatgagca gctgtccttc cagggttcac aagtagtggt gccttcttnc 60tgtccctccg atggagatta tctgcatgtc gtggtcgtgt cctgatcgag tcgtcgttga 120gtccctatgt tttttcttca agaaatgtga gtcctatgtc agtctggttg cgtttgtgaa 180cattttctgc tgctgcgcag cagtttggtt ggaactgtgc aatgaaataa attgaaccct 240ggtttctggt tatgtgtgtt agctaatgtt tttgaagtgg aagctntaat cttntatcgc 300gttgctacta caattctgnt tgtgttttga tgttcttgtt tct 343 5 5512 DNA SaccharumHybrid Cultivar H32-8560 misc_feature (2218) The “n” at position 2218 isany nucleotide. 5 gaattcatta tgtggtctag gtaggttcta tatataagaa aacttgaaatgttctaaaaa 60 aaaattcaag cccatgcatg attgaagcaa acggtatagc aacggtgttaacctgatcta 120 gtgatctctt gcaatcctta acggccacct accgcaggta gcaaacggcgtccccctcct 180 cgatatctcc gcggcgacct ctggcttttt ccgcggaatt gcgcggtggggacggattcc 240 acaaccgcga cgcaaccgcc tctcgccgct gggccccaca ccgctcggtgccgtagcctc 300 acgggactct ttctccctcc tcccccgtta taaattggct tcatcccctccttgcctcat 360 ccatccaaat cccagtcccc aatcccatcc cttcgtcgga gaaattcatcgaagcgaagc 420 gaatcctcgc gatcctctca aggtactgcg agttttcgat ccccctctcgacccctcgta 480 tgtttgtgtt tgtcgtacgt ttgattaggt atgctttccc tgtttgtgttcgtcgtagcg 540 tttgattagg tatgctttcc ctgttcgtgt tcatcgtagt gtttgattaggtcgtgtgag 600 gcgatggcct gctcgcgtcc ttcgatctgt agtcgatttg cgggtcgtggtgtagatctg 660 cgggctgtga tgaagttatt tggtgtgatc tgctcgcctg attctgcgggttggctcgag 720 tagatatgga tggttggacc ggttggttcg tttaccgcgc tagggttgggctgggatgat 780 gttgcatgcg ccgttgcgcg tgatcccgca gcaggacttg cgtttgattgccagatctcg 840 ttacgattat gtgatttggt ttggacttat tagatctgta gcttctgcttatgttgccag 900 atgcgcctac tgctccatat gcctgatgat aatccataaa tggcagtggaaatcaactag 960 ttgattgcgg agtcatgtat cagctacagg tgtagggact agctacaggtgtagggactg 1020 cgtctaattg tttggtcctt aactcatgtg caattatgca atttagtttagatgtttgtt 1080 ccaatcatct aggctgtaaa agggacactg gttagattgc tgtttaatctttttagtaga 1140 ttatattata ttggtaactt attaacccta ttacatgcca taacgtggattctgctcatg 1200 cctgatgata atcatagatc actgtggaat taattagttg attgttgaatcatgtttcat 1260 gtacatacca cggcacaatt gcttagttcc ttaacaaatg caaattttactgatccatgt 1320 atgatttgcg tggttctcta atgtgaaata ctatagctac ttgttagtaagaatcaggtt 1380 cgtatgctta atgctgtatg tgccttctgc tcatgcctga tgataatcatatatcactgg 1440 aattaattag ttgatcgttt aatcatatat caagtacata ccatggcacaatttttagtc 1500 acttaaccca tgcagattga actggtccct gcatgttttg ctaaattgttctattctgat 1560 tagaccatat atcaggtatt tttttttggt aatggttctc ttattttaaatgctatatag 1620 ttctggtact tgttagaaag atctggttca tagtttagtt gcctatccttcgaattagga 1680 tgctgagcag ctgatcctat agctttgttt catgtatcaa ttcttttgtgttcaacagtc 1740 agtttttgtt agattcattg taacttatgt tcgcttactc ttctggtcctcaatgcttgc 1800 agatgcagat cttcgttaag accctcactg gcaagaccat cacccttgaggttgagtctt 1860 cagacamtat tgacmatgtc maggctaaga tacaggacaa ggaaggcattcctccggatc 1920 agcagaggct gatctttgct ggcaagcagc tcgaggatgg ccgtaccctagytgactaca 1980 acatccagaa ggagtccacc stccacctgg tgctcaggct caggggaggcatgcaaatct 2040 tcgtcaagac cctcactggc aagactatca cgcttgaggt cgagtcttctgacacgatcg 2100 acaacgtgaa ggccaagatc caggacaagg agggaatccc cccggaccagcagcgtctca 2160 tcttcgctgg caagcagctc gaggatggcc gcaccctcgc tgactacaacatccagangg 2220 agtcgantnt ccaccttgtg ctcaggttna ggggtggcat gcagatttttgtcaagacct 2280 tnactggcaa gaccatcacc ttggaggtgg agtcttcgga caccatngacaatgtgaagg 2340 ngaagatcca ggacaaggaa ggaatccccc cagaccagca gcgtcttatttttgctggca 2400 agcagcttga ggatggccgc accctagcag actacaacat ccagaaggagtccacccttc 2460 acctggtgct ccgcttncgc ggtggtatgc agatcttcgt caagaccctcaccggcaaga 2520 ccatcaccct ggaggtggag tcctctgaca ccatcgacaa tgtgaaggcgaagatccagg 2580 acaaggaggg catccccccg gaccagcagc gtctcatctt cgccggcaagcagctggagg 2640 atggccgcac cctggcagac tacaacatcc agaaggagtc cactctccacctggtgctcc 2700 gtctccgtgg tggccagtaa tcctgggcca tgaagctgtc cttccaggttcacaagtctg 2760 gtgccttctt ctgtccctcc gatggagatt atctgcatgt cgtggtcgtgtcctgatcga 2820 atcctcgttg aatccctatg tttttcttca agaaatgtga gtcctatgtcagtctggttg 2880 cgtttgtgaa catttctgct gctgagcagc actttggctg gaactgtgcaatgaaataaa 2940 tggaaccctg gtttctggtt atgtgtgtgt tagctaatgt ttttgaagtggaagctctaa 3000 tcttctatcg cgttgctact acaattctgc ttgtgttttg atgttcttggtttctgttag 3060 ttggttcaga ggaagttttg cttccacaga ctaagatgca gttgaactttggttgccctg 3120 gtttctagat ttcatttgtg ctggttgagt gatagtaaga aacaaccggtgttcacatat 3180 aatcaggttt tgtgctgctc gagtgatcgt caaaaaccac cggtgttcacatctaaaaag 3240 gtttcgatcc ccaggtttag atctcccgtt taattccaaa aaaaaagttctgtgtacttg 3300 catttagttg ggtggttgat gctggaaaga gtaactttca agagtaataatctttggtga 3360 ctactctgtt tcaactgatc aatccctagg aaaggtacac ctttacttagggaagaaatt 3420 cttagaacct tgcactttgt ttcaactgat aatagtatac tttattagataaaaaatatt 3480 cagatatatt agacaccgga tgtcatccac tcatccttac aaacctctgtcatggtcctg 3540 cagaaatgtt tgccagctcc agtggcttcc tgataaatct gtggagtgcctgttaatcgg 3600 ctgccaattt ttgctgagca ctgtatatat gttagtaagt actattgggccaccaattcg 3660 attttgacac agcactattg gtccaccaat tcgattctga cacagcactgcataatttga 3720 aacgtgttgc tccattttgc aaggctacaa atttagatca tgtttagcattctgtgggat 3780 acaatatatg gatatcgaac aaacttggta tgtcagagaa aaaatagtttattttcaaaa 3840 ctaacatttt taaagccttc tatgaacttt aaaccttcag catttgggatcaagatgagt 3900 gctcgaacaa gagtgcactt tttctccaaa ataatctact acagagttcttttttatata 3960 taaaaaaact tatacttaac agataaatca gactttttct gctccatatcaccttgacaa 4020 atcaaagaag cagcaccagc gaagggtatt attattgagg taaatataagatctcgttta 4080 ctgaaaaaga ccgcgtgttt acctaaacta ccattttgct ttgatagcagcatacatgtg 4140 atagaattgc ggatcctacc gtgctgactg tgaaggtggt aggggtgagagattggtggg 4200 cgaggtctga acgagcgaga acagtactgc atttactgtt cacaaggaggcggcttaggt 4260 tttgggtctc ccagctctct aagggaagct gagaattatg attctcttgcttaattattt 4320 cttaaccaaa gttataaata tatagcctat gagatcctaa tttatggaaataactaaact 4380 attttaagga aatatataaa tagataatca gcccactaac gggcctagcgcccactaaca 4440 ggcctggtgc tgagcccgac ataacatctc tccccgcctg gagaaacagctcgtcctcga 4500 gctgaaatct ggtagaagca tcatcaacca acaccggggt catgctggaacactgcatca 4560 ggcgctaccg cagctggtac gtcgtcgtcg aggaagtcag ccgactccaagtagaacagt 4620 cgcttacact gatgtccgcg gacgtagggc tcatcacaat tgtaacaaagcccttgacga 4680 cggcactcca acagctcctc cggtgtgaga cgacgaaacg agggagctagagcgggtagt 4740 ggcgcgggaa cagccagtgt agcgcctgta gtcaccgagg gatggggcggtcgggcgccg 4800 cgaggctgcg gtgcaggtgg aggtttcaca ttcctcaaac gcccgtgccaagtacatggc 4860 ggactggagg tcgggcggtg cgcggagctg aacctgctta cggaggtggtccggcagccc 4920 acccacgtac aactccgcct tttggcgagc ggagaggttg tgggcatggcacaggacggc 4980 gttgtaacgc tccgagtaat cctgaacgga agaaccaaaa ggaaggcgggcaagctccgc 5040 caaccgagtg cccaaaacag gaggcccgaa gcgaagcgag cataattcgcggaagcgctc 5100 ccaaggaggc ataccctcgt cttgctccag ggcgtagtac catgtctgggcaacaccccg 5160 aagatggtag gacgcgagcc atgtgcgagc ggaggcgagc gtctgctggccgcggaagaa 5220 ctgctcgcac tggttcaacc aattcagggg atcggtcgaa ccgtcgtacgtagggaactc 5280 cagtttgtag aatttgggcc ccgcctgggc gcctgcgagg gcagcagcaagggctgggtc 5340 gagccccccc tgcggctggc cgcccgaggg agagggcgcc cgaagaacagcggcccgtcc 5400 acccccccga aaagagtgct ggcggggggt agggaagaca tcgttgtcgccgccgccgtc 5460 gtgtaggctg tcggggaagg cgacgtggcc atcgagtaga tccggggaattc 5512 6 305 PRT Saccharum Hybrid Cultivar H32-8560 VARIANT (22) The“Xaa” at position 22 is any amino acid. 6 Met Gln Ile Phe Val Lys ThrLeu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Ser Ser Asp XaaIle Asp Xaa Val Xaa Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro ProAsp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly ArgThr Leu Xaa Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Xaa His Leu ValLeu Arg Leu Arg Gly Gly Met Gln Ile Phe 65 70 75 80 Val Lys Thr Leu ThrGly Lys Thr Ile Thr Leu Glu Val Glu Ser Ser 85 90 95 Asp Thr Ile Asp AsnVal Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp GlnGln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg ThrLeu Ala Asp Tyr Asn Ile Gln Xaa Glu Ser Xaa Xaa His 130 135 140 Leu ValLeu Arg Xaa Arg Gly Gly Met Gln Ile Phe Val Lys Thr Xaa 145 150 155 160Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Ser Ser Asp Thr Xaa Asp 165 170175 Asn Val Lys Xaa Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln 180185 190 Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu195 200 205 Ala Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val LeuArg 210 215 220 Xaa Arg Gly Gly Met Gln Ile Phe Val Lys Thr Leu Thr GlyLys Thr 225 230 235 240 Ile Thr Leu Glu Val Glu Ser Ser Asp Thr Ile AspAsn Val Lys Ala 245 250 255 Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro AspGln Gln Arg Leu Ile 260 265 270 Phe Ala Gly Lys Gln Leu Glu Asp Gly ArgThr Leu Ala Asp Tyr Asn 275 280 285 Ile Gln Lys Glu Ser Thr Leu His LeuVal Leu Arg Leu Arg Gly Gly 290 295 300 Gln 305 7 1802 DNA SaccharumHybrid Cultivar H32-8560 7 gaattcatta tgtggtctag gtaggttcta tatataagaaaacttgaaat gttctaaaaa 60 aaaattcaag cccatgcatg attgaagcaa acggtatagcaacggtgtta acctgatcta 120 gtgatctctt gcaatcctta acggccacct accgcaggtagcaaacggcg tccccctcct 180 cgatatctcc gcggcgacct ctggcttttt ccgcggaattgcgcggtggg gacggattcc 240 acaaccgcga cgcaaccgcc tctcgccgct gggccccacaccgctcggtg ccgtagcctc 300 acgggactct ttctccctcc tcccccgtta taaattggcttcatcccctc cttgcctcat 360 ccatccaaat cccagtcccc aatcccatcc cttcgtcggagaaattcatc gaagcgaagc 420 gaatcctcgc gatcctctca aggtactgcg agttttcgatccccctctcg acccctcgta 480 tgtttgtgtt tgtcgtacgt ttgattaggt atgctttccctgtttgtgtt cgtcgtagcg 540 tttgattagg tatgctttcc ctgttcgtgt tcatcgtagtgtttgattag gtcgtgtgag 600 gcgatggcct gctcgcgtcc ttcgatctgt agtcgatttgcgggtcgtgg tgtagatctg 660 cgggctgtga tgaagttatt tggtgtgatc tgctcgcctgattctgcggg ttggctcgag 720 tagatatgga tggttggacc ggttggttcg tttaccgcgctagggttggg ctgggatgat 780 gttgcatgcg ccgttgcgcg tgatcccgca gcaggacttgcgtttgattg ccagatctcg 840 ttacgattat gtgatttggt ttggacttat tagatctgtagcttctgctt atgttgccag 900 atgcgcctac tgctccatat gcctgatgat aatccataaatggcagtgga aatcaactag 960 ttgattgcgg agtcatgtat cagctacagg tgtagggactagctacaggt gtagggactg 1020 cgtctaattg tttggtcctt aactcatgtg caattatgcaatttagttta gatgtttgtt 1080 ccaatcatct aggctgtaaa agggacactg gttagattgctgtttaatct ttttagtaga 1140 ttatattata ttggtaactt attaacccta ttacatgccataacgtggat tctgctcatg 1200 cctgatgata atcatagatc actgtggaat taattagttgattgttgaat catgtttcat 1260 gtacatacca cggcacaatt gcttagttcc ttaacaaatgcaaattttac tgatccatgt 1320 atgatttgcg tggttctcta atgtgaaata ctatagctacttgttagtaa gaatcaggtt 1380 cgtatgctta atgctgtatg tgccttctgc tcatgcctgatgataatcat atatcactgg 1440 aattaattag ttgatcgttt aatcatatat caagtacataccatggcaca atttttagtc 1500 acttaaccca tgcagattga actggtccct gcatgttttgctaaattgtt ctattctgat 1560 tagaccatat atcaggtatt tttttttggt aatggttctcttattttaaa tgctatatag 1620 ttctggtact tgttagaaag atctggttca tagtttagttgcctatcctt cgaattagga 1680 tgctgagcag ctgatcctat agctttgttt catgtatcaattcttttgtg ttcaacagtc 1740 agtttttgtt agattcattg taacttatgt tcgcttactcttctggtcct caatgcttgc 1800 ag 1802 8 5174 DNA Saccharum Hybrid CultivarH32-8560 misc_feature (9) The “n” at position 9 is any nucleotide. 8aagttttgnt aaaatgaaca aagaattggg gaaactatag ccaaagtggg tggggaatgg 60tgccaaacaa aacttcgtaa accaacccaa aaagatccgg aaaacaaatg gatacgtgca 120gggcatgcat gcaatagccc agccataaaa agcggcgagc caatgcccgg gtgtcaaaca 180aaatggcgcc tgtgccggct ctggctgctt ccggctcagc tttcggaacg atccgccgca 240gtttggcctc gcatatgatg acgatgatgg tctcctcttc tcgatttgta gctccggcat 300gggagccacc tcctgtcggc tcacacatag cacgcgcctt agcccgtgct cgctctcccc 360tagatgcttc acctgcgcca atcagtgtga gcccatcgtg tcagatggta ctcgtacgta 420tggagtaacg tgataccaca acacgtacac tggtcagaat tgatagtata tgatcctgtc 480gacccgatgt gttttagtac cttgcagtgg ccggagagga gtggccgcgc gcatgcggcg 540caggggttct ccgcgctcgc tgatcgcttc ctcactgtgc gctcgtttag gaacaccacc 600tcgtggtcgc tcaccatgtg tgactgcatg caacgctacg aatcaggacc cagatggaaa 660cgaagcgcct ctcgaccacc tctgcctcgg tgatggttgg tgtgcagtgc gtacgcatgc 720acgctaccaa tatcatacct ggatgccggt gcaatcgaac agcttcaggt tgtcgacgcg 780gacggcgaag caggacgcgt acttccatat ctttgggttc cattacgtac cgtcaatcga 840ataaataaag agaagagttt gagatcagct tgttgggagc aggtgaccgc ccgacatgca 900tgccgattgt cgacggcacg gaaataaaca acacatttgt gagggagcca gggaggcagt 960ggcggcacag cgtcgcggca cagtcgatgc agaagtggtt cttgtcgttc ttgcgctccc 1020cccgggtgtg cagcgcacgc ctttgaaaaa ctccgatagc aggccacaca gccattgcgg 1080ggcgccgcgc acggccgcca gctgcatccc cgtttgttcg cacatgcgct aggtggtcct 1140gcggccgttc cttgcaccgc ggagacgcgg ggtggaccag tgggggaatg gatgaactgc 1200tggtaggttt ggttggattg gcgagtgcgt agagggggca tgggcaacga tagactcgat 1260tcaattcaaa gactgaaaat agtggagttc taacaccatt ctgtgcggcg ctaattctcg 1320acatggcagg cgtaagcata ataccgacat ggcatgcaac gatgttcgtg aacagtggtg 1380acacatggat atggtggccg tccaggggat tcgttccatt caattcaaag accgaaaatc 1440gcggggttcc gtagcatttt gtgcggtgct aattctcgaa catgcgagac gtaagcctaa 1500taccgagatg gcatgcaaca atgttcgtga acaacagtga cacgtggatg cggtggccgt 1560ctagggattc gcgttctaag ctggtatatg tgcggtgtta attcttgaca tgcggggcgt 1620aagtgtaata ccaagatgaa cggtgacacg tggacgcggg ggtcgtcaaa caattcattc 1680cgtggtctag ggtaggttat atataaaggc cagtcttagt gggggatttt atggccatgt 1740tattaatgca acccatattt ggaaaacagt gcaggaagag tttcatcttc gtaaaactct 1800ctctaattcc atgaaactct tatcatctct ctcttcatca atacggtgcc acatcagcct 1860atttaatgtc catgaaactc tgatgaaatc cactgagacg ggcctcagaa aacttgaaat 1920cttctaaaaa aaattcaagt ccatgcatga ttgaagcaaa cggtatagca acggtgttaa 1980cctgatctag tgatctcttg taatccttaa cggccaccta ccacaggtag caaacggcgt 2040ccccctcctc gatatctccg cggcggcctc tggctttttc cgcggaattg cgcggtgggg 2100acggattcct cgagaccgcg acacaaccgc ctttcgccgc tgggccccac accgctcggt 2160gccgtagcct cacgggactc tttctccctc ctcccccgct ataaattggc ttcatcccct 2220ccttgcctca tccatccaaa tcccagtccc caatcccagc ccatcgtcgg agaaattcat 2280agaagcgaag cgaatcctcg cgatcctctc aaggtagtgc gagttttcga ttcccctctc 2340gacccctcgt atgctttccc tgtttgtgtt tcgtcgtagc gtttgattag gtatgctttc 2400cctgtttgtg ttcgtcgtag cgtttgattt ggtatgcttt ccccgttcgt gttcctcgta 2460gtgtttgatt aggtcgtgtg aggcgatggc ctgctcgcat ccttcgatct gtagtcgatt 2520tgcgggtcgt ggtgtagatc tgcgggctgt gatgaagtta tttggtgtga tcgtgctcgc 2580ctgattctgc gggttggctc gagtagatat gatggttgga ccggttggtt tgtttaccgc 2640gctagggttg ggctgggatg atgttgcatg cgccgttgcg cgtgatcccg cagcaggact 2700tgcgtttgat tgccagatct cgttacgatt atgtgatttg gtttggactt tttagatctg 2760tagcttctgc ttatgtgcca gatgcgccta ctgctcatat gcctgatgat aatcataaat 2820ggctgtggaa ctaactagtt gattgcggag tcatgtatca gctacaggtg tagggactag 2880ctacaggtgt agggacttgc gtctaaattg tttggtcctg tactcatgtt gcaattatgc 2940aatttagttt agattgtttg ttccactcat ctaggctgta aaagggacac tgcttagatt 3000gctgtttaat ctttttagta gattatatat tatattggta acttattacc cttattacat 3060gccatacgtg acttctgctc atgcctgatg ataatcatag atcactgtgg aattaattag 3120ttgattgttg aatcatgttt catgtacata ccacggcaca attgcttagt tccttaacaa 3180atgcaaattt tactgatcca tgtatgattt gcgtggttct ctaatgtgaa atactatagc 3240tacttgttag taagaatcag gttcgtatgc ttaatgctgt atgtgccttc tgctcatgcc 3300tgatgataat catatatcac tggaattaat tagttgatcg tttaatcata tatcaagtac 3360ataccatggc acaattttta gtcacttaac ccatgcagat tgaactggtc cctgcatgtt 3420ttgctaaatt gttctatttc tgattagacc atatatcatg taattttttt tttgggtaat 3480ggttctccta ttttaaatgc tatatagttc tggtacttgt tagaaaaatc tgcttccata 3540gtttagttgc ttatccctcg aattatgatg ctgagcagct gatcctatag ctttgtttca 3600kgtatcaatt cttgtgttca acagtcagtt tttgttagat tcattgtaac ttatggtcgc 3660ttactcttct ggtcctcaat gcttgcagat gcagattttc gttaagaccc tcactggcaa 3720gaccatcacc cttgaggttg agtcctcaga cactattgac aatgtcaagg ctaagatcca 3780ggacaaggaa ggcattcctc cagatcagca gaggctgaty tttgctggca agcagctcga 3840ggatggccgt accctagctg actacaacat ccagaaggag tccaccctcc acctggtgct 3900caggcttagg ggaggcatgc agattttcgt caagaccctc actggcaaga ctatcacgct 3960tgaggtcgag tcttctgaca cgatcgacaa cgtgaaggcc aagatccagg acaaggaggg 4020aatccccccg gaccagcagc gtytcatttt cgctggcaag cagctcgagg atggccgcac 4080cctcgctgac tacaacatcc agaaggagtc gactctccac cttgtgctca ggctcagggg 4140tggcatgcag atcttcgtca agaccctcac tggcaagacc atcaccttgg aggtggagtc 4200ctcggacacc attgacaatg tgaaggcgaa gatccaggac aaggagggca tccccccgga 4260ccagcagcgt ctcatyttcg ccggcaagca rcttgaggat ggccgcaccc ttgcgganta 4320caacatccag aargagtcca cccttcacct ggtgctccgc cttcgtggtg gtatgcagat 4380tttcgtcaag accctcaccg gcaagaccat caccctggag gtggagtcct ctgacaccat 4440tgacaatgtg aaggcgaaga tccaggataa ggagggcatc cccccggacc agcagcgtyt 4500tatctttgct ggcaagcagc ttgaggatgg ccgcaccctg gcagantaca acatccagaa 4560ggagtccacc cttcacctgg tgctccgcct tcgcggtggt atgcagatyt tcgtcaagac 4620cctcaccggc aagaccatca ccctggaggt ggagtcctct gacaccatcg acaatgtgaa 4680ggcgaagatc caggacaagg agggcatccc cccggaccag cagcgtctca tcttcgccgg 4740caagcagctg gaggatggcc gcaccctggc agactacaac atccagaagg agtccactct 4800ccacctggtg ctccgtctcc gtggtggcca gtaagtcctg ggccatgagc agctgtcctt 4860ccagggttca caagtagtgg tgccttcttn ctgtccctcc gatggagatt atctgcatgt 4920cgtggtcgtg tcctgatcga gtcgtcgttg agtccctatg ttttttcttc aagaaatgtg 4980agtcctatgt cagtctggtt gcgtttgtga acattttctg ctgctgcgca gcagtttggt 5040tggaactgtg caatgaaata aattgaaccc tggtttctgg ttatgtgtgt tagctaatgt 5100ttttgaagtg gaagctntaa tcttntatcg cgttgctact acaattctgn ttgtgttttg 5160atgttcttgt ttct 5174 9 381 PRT Saccharum Hybrid Cultivar H32-8560VARIANT (119) The “Xaa” at position 119 is any amino acid. 9 Met Gln IlePhe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val GluSer Ser Asp Thr Ile Asp Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys GluGly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln LeuGlu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser ThrLeu His Leu Val Leu Arg Leu Arg Gly Gly Met Gln Ile Phe 65 70 75 80 ValLys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Ser Ser 85 90 95 AspThr Ile Asp Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110Pro Pro Asp Gln Gln Arg Xaa Ile Phe Ala Gly Lys Gln Leu Glu Asp 115 120125 Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His 130135 140 Leu Val Leu Arg Leu Arg Gly Gly Met Gln Ile Phe Val Lys Thr Leu145 150 155 160 Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Ser Ser Asp ThrIle Asp 165 170 175 Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile ProPro Asp Gln 180 185 190 Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu AspGly Arg Thr Leu 195 200 205 Ala Xaa Tyr Asn Ile Gln Lys Glu Ser Thr LeuHis Leu Val Leu Arg 210 215 220 Leu Arg Gly Gly Met Gln Ile Phe Val LysThr Leu Thr Gly Lys Thr 225 230 235 240 Ile Thr Leu Glu Val Glu Ser SerAsp Thr Ile Asp Asn Val Lys Ala 245 250 255 Lys Ile Gln Asp Lys Glu GlyIle Pro Pro Asp Gln Gln Arg Xaa Ile 260 265 270 Phe Ala Gly Lys Gln LeuGlu Asp Gly Arg Thr Leu Ala Xaa Tyr Asn 275 280 285 Ile Gln Lys Glu SerThr Leu His Leu Val Leu Arg Leu Arg Gly Gly 290 295 300 Met Gln Ile PheVal Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 305 310 315 320 Val GluSer Ser Asp Thr Ile Asp Asn Val Lys Ala Lys Ile Gln Asp 325 330 335 LysGlu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 340 345 350Gln Leu Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys Glu 355 360365 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Gln 370 375 380 103688 DNA Saccharum Hybrid Cultivar 32-8560 misc_feature (9) The “n” atposition 9 is any nucleotide. 10 aagttttgnt aaaatgaaca aagaattggggaaactatag ccaaagtggg tggggaatgg 60 tgccaaacaa aacttcgtaa accaacccaaaaagatccgg aaaacaaatg gatacgtgca 120 gggcatgcat gcaatagccc agccataaaaagcggcgagc caatgcccgg gtgtcaaaca 180 aaatggcgcc tgtgccggct ctggctgcttccggctcagc tttcggaacg atccgccgca 240 gtttggcctc gcatatgatg acgatgatggtctcctcttc tcgatttgta gctccggcat 300 gggagccacc tcctgtcggc tcacacatagcacgcgcctt agcccgtgct cgctctcccc 360 tagatgcttc acctgcgcca atcagtgtgagcccatcgtg tcagatggta ctcgtacgta 420 tggagtaacg tgataccaca acacgtacactggtcagaat tgatagtata tgatcctgtc 480 gacccgatgt gttttagtac cttgcagtggccggagagga gtggccgcgc gcatgcggcg 540 caggggttct ccgcgctcgc tgatcgcttcctcactgtgc gctcgtttag gaacaccacc 600 tcgtggtcgc tcaccatgtg tgactgcatgcaacgctacg aatcaggacc cagatggaaa 660 cgaagcgcct ctcgaccacc tctgcctcggtgatggttgg tgtgcagtgc gtacgcatgc 720 acgctaccaa tatcatacct ggatgccggtgcaatcgaac agcttcaggt tgtcgacgcg 780 gacggcgaag caggacgcgt acttccatatctttgggttc cattacgtac cgtcaatcga 840 ataaataaag agaagagttt gagatcagcttgttgggagc aggtgaccgc ccgacatgca 900 tgccgattgt cgacggcacg gaaataaacaacacatttgt gagggagcca gggaggcagt 960 ggcggcacag cgtcgcggca cagtcgatgcagaagtggtt cttgtcgttc ttgcgctccc 1020 cccgggtgtg cagcgcacgc ctttgaaaaactccgatagc aggccacaca gccattgcgg 1080 ggcgccgcgc acggccgcca gctgcatccccgtttgttcg cacatgcgct aggtggtcct 1140 gcggccgttc cttgcaccgc ggagacgcggggtggaccag tgggggaatg gatgaactgc 1200 tggtaggttt ggttggattg gcgagtgcgtagagggggca tgggcaacga tagactcgat 1260 tcaattcaaa gactgaaaat agtggagttctaacaccatt ctgtgcggcg ctaattctcg 1320 acatggcagg cgtaagcata ataccgacatggcatgcaac gatgttcgtg aacagtggtg 1380 acacatggat atggtggccg tccaggggattcgttccatt caattcaaag accgaaaatc 1440 gcggggttcc gtagcatttt gtgcggtgctaattctcgaa catgcgagac gtaagcctaa 1500 taccgagatg gcatgcaaca atgttcgtgaacaacagtga cacgtggatg cggtggccgt 1560 ctagggattc gcgttctaag ctggtatatgtgcggtgtta attcttgaca tgcggggcgt 1620 aagtgtaata ccaagatgaa cggtgacacgtggacgcggg ggtcgtcaaa caattcattc 1680 cgtggtctag ggtaggttat atataaaggccagtcttagt gggggatttt atggccatgt 1740 tattaatgca acccatattt ggaaaacagtgcaggaagag tttcatcttc gtaaaactct 1800 ctctaattcc atgaaactct tatcatctctctcttcatca atacggtgcc acatcagcct 1860 atttaatgtc catgaaactc tgatgaaatccactgagacg ggcctcagaa aacttgaaat 1920 cttctaaaaa aaattcaagt ccatgcatgattgaagcaaa cggtatagca acggtgttaa 1980 cctgatctag tgatctcttg taatccttaacggccaccta ccacaggtag caaacggcgt 2040 ccccctcctc gatatctccg cggcggcctctggctttttc cgcggaattg cgcggtgggg 2100 acggattcct cgagaccgcg acacaaccgcctttcgccgc tgggccccac accgctcggt 2160 gccgtagcct cacgggactc tttctccctcctcccccgct ataaattggc ttcatcccct 2220 ccttgcctca tccatccaaa tcccagtccccaatcccagc ccatcgtcgg agaaattcat 2280 agaagcgaag cgaatcctcg cgatcctctcaaggtagtgc gagttttcga ttcccctctc 2340 gacccctcgt atgctttccc tgtttgtgtttcgtcgtagc gtttgattag gtatgctttc 2400 cctgtttgtg ttcgtcgtag cgtttgatttggtatgcttt ccccgttcgt gttcctcgta 2460 gtgtttgatt aggtcgtgtg aggcgatggcctgctcgcat ccttcgatct gtagtcgatt 2520 tgcgggtcgt ggtgtagatc tgcgggctgtgatgaagtta tttggtgtga tcgtgctcgc 2580 ctgattctgc gggttggctc gagtagatatgatggttgga ccggttggtt tgtttaccgc 2640 gctagggttg ggctgggatg atgttgcatgcgccgttgcg cgtgatcccg cagcaggact 2700 tgcgtttgat tgccagatct cgttacgattatgtgatttg gtttggactt tttagatctg 2760 tagcttctgc ttatgtgcca gatgcgcctactgctcatat gcctgatgat aatcataaat 2820 ggctgtggaa ctaactagtt gattgcggagtcatgtatca gctacaggtg tagggactag 2880 ctacaggtgt agggacttgc gtctaaattgtttggtcctg tactcatgtt gcaattatgc 2940 aatttagttt agattgtttg ttccactcatctaggctgta aaagggacac tgcttagatt 3000 gctgtttaat ctttttagta gattatatattatattggta acttattacc cttattacat 3060 gccatacgtg acttctgctc atgcctgatgataatcatag atcactgtgg aattaattag 3120 ttgattgttg aatcatgttt catgtacataccacggcaca attgcttagt tccttaacaa 3180 atgcaaattt tactgatcca tgtatgatttgcgtggttct ctaatgtgaa atactatagc 3240 tacttgttag taagaatcag gttcgtatgcttaatgctgt atgtgccttc tgctcatgcc 3300 tgatgataat catatatcac tggaattaattagttgatcg tttaatcata tatcaagtac 3360 ataccatggc acaattttta gtcacttaacccatgcagat tgaactggtc cctgcatgtt 3420 ttgctaaatt gttctatttc tgattagaccatatatcatg taattttttt tttgggtaat 3480 ggttctccta ttttaaatgc tatatagttctggtacttgt tagaaaaatc tgcttccata 3540 gtttagttgc ttatccctcg aattatgatgctgagcagct gatcctatag ctttgtttca 3600 kgtatcaatt cttgtgttca acagtcagtttttgttagat tcattgtaac ttatggtcgc 3660 ttactcttct ggtcctcaat gcttgcag3688 11 2146 DNA Lycopersicon esculentum misc_feature (16) The “n” atposition 16 is any nucleotide. 11 agatctacaa ttatcngcaa cgtgttacacattttgtgct acaatatacc ttcaccattt 60 tgtgtatata taaaggttgc atctcttcaaacaaaaatca ctccatcaca acacaatgtc 120 ttcttcttct tctattacta ctactcttcctttatgcacc aacaaatccc tctcttcttc 180 cttcaccacc accaactcat ccttgttatcaaaaccctct caacttttcc tccacggaag 240 gcgtaatcaa agtttcaagg tttcatgcaacgcaaacaac gttgacaaaa accctgacgc 300 tgttgataga cgaaacgttc ttttagggttaggaggtctt tatggtgcag ctaatcttgc 360 accattagcg actgctgcac ctataccacctcctgatctc aagtcttgtg gtactgccca 420 tgtaaaagaa ggtgttgatg taatatacagttgttgccct cctgtacccg atgatatcga 480 tagtgttccg tactacaagt tcccttctatgactaaactc cgcatccgcc cccctgctca 540 tgcggcggat gaggagtacg tagccaagtatcaattggct acgagtcgaa tgagggaact 600 tgataaagac ccctttgacc ctcttggctttaaacaacaa gctaatattc attgtgctta 660 ttgcaacggt gcttacaaag ttggtggcaaagaattgcaa gttcatttct cgtggctttt 720 ctttcccttt catagatggt acttgtacttttacgaaaga attttgggat cacttattaa 780 tgatccaact tttgctttac cttactggaattgggatcat ccaaaaggca tgcgtatacc 840 tcccatgttt gatcgtgagg gatcatctctttacgatgag aaacgtaacc aaaatcatcg 900 caatggaact attattgatc ttggtcattttggtaaggaa gttgacacac ctcagctaca 960 gataatgact aataatttaa ccctaatgtaccgtcaaatg gttactaatg ctccttgccc 1020 ttcccaattc ttcggtgctg cttacctctgggttctgaac ccaagtccgg gtcagggtac 1080 tattgaaaac atccctcata ctccggttcacatctggacc ggtgacaaac ctcgtcaaaa 1140 aaacggtgaa gacatgggta atttctactcagccggttta gatccgattt tttactgcca 1200 ccatgccaat gtggacagga tgtggaatgaatggaaatta attggcggga aaagaaggga 1260 tttaacagat aaagattggt tgaactctgaattctttttc tacgatgaaa atcgtaaccc 1320 ttaccgtgtg aaagtccgtg atgttttggacagtaaaaaa atgggattcg attacgcgcc 1380 aatgcccact ccatggcgta attttaaaccaatcagaaag tcatcatcag gaaaagtgaa 1440 tacagcgtca attgcaccag ttagcaaggtgttcccattg gcgaagctgg accgtgcgat 1500 ttcgttctct atcacgcggc cagcctcgtcaaggacaaca caagagaaaa atgagcagga 1560 ggagattctg acattcaata aaatatcgtatgatgatagg aactatgtaa ggttcgatgt 1620 gtttctgaac gtggacaaga ctgtgaatgcagatgagctt gataaggcgg agtttgcagg 1680 gagttatact agcttgccgc atgttcatggaagtaatact aatcatgtta ccagtgttac 1740 tttcaagctg gcgataactg aactgttggaggatattgga ttggaagatg aagatactat 1800 cgcggtgact ttaattccaa aagctggcggtgaaggtgta tccattgaaa gtgtggagat 1860 caagcttgag gattgttaaa gtctgcatgagttggtggct atggagccaa atttatgttt 1920 aattagtata attatgtgtg gtttgagttatgttttatgt taaaatgtat cagctcgatc 1980 gatagctgat tgctagttgt gttaatgctatgtatgaaat aaataaatgg ttgtcttcca 2040 ttcagtttat cattttttgt cattctaattaacggttaac ttttttttct actatttata 2100 cgaagctact atactatgta tatcatttggaaaattatat attatt 2146 12 3509 DNA Zea mays 12 gaattccggc gtgggcgctgggctagtgct cccgcagcga gcgatctgag agaacggtag 60 agttccggcc gggcgcgcgggagaggagga gggtcgggcg gggaggatcc gatggccggg 120 aacgagtgga tcaatgggtacctggaggcg atcctcgaca gccacacctc gtcgcggggt 180 gccggcggcg gcggcggcgggggggacccc aggtcgccga cgaaggcggc gagcccccgc 240 ggcgcgcaca tgaacttcaacccctcgcac tacttcgtcg aggaggtggt caagggcgtc 300 gacgagagcg acctccaccggacgtggatc aaggtcgtcg ccacccgcaa cgcccgcgag 360 cgcagcacca ggctcgagaacatgtgctgg cggatctggc acctcgcgcg caagaagaag 420 cagctggagc tggagggcatccagagaatc tcggcaagaa ggaaggaaca ggagcaggtg 480 cgtcgtgagg cgacggaggacctggccgag gatctgtcag aaggcgagaa gggagacacc 540 atcggcgagc ttgcgccggttgagacgacc aagaagaagt tccagaggaa cttctctgac 600 cttaccgtct ggtctgacgacaataaggag aagaagcttt acattgtgct catcagcgtg 660 catggtcttg ttcgtggagaaaacatggaa ctaggtcgtg attctgatac aggtggccag 720 gtgaaatatg tggtcgaacttgcaagagcg atgtcaatga tgcctggagt gtacagggtg 780 gacctcttca ctcgtcaagtgtcatctcct gacgtggact ggagctacgg tgagccaacc 840 gagatgttat gcgccggttccaatgatgga gaggggatgg gtgagagtgg cggagcctac 900 attgtgcgca taccgtgtgggccgcgggat aaatacctca agaaggaagc gttgtggcct 960 tacctccaag agtttgtcgatggagccctt gcgcatatcc tgaacatgtc caaggctctg 1020 ggagagcagg ttggaaatgggaggccagta ctgccttacg tgatacatgg gcactatgcc 1080 gatgctggag atgttgctgctctcctttct ggtgcgctga atgtgccaat ggtgctcact 1140 ggccactcac ttgggaggaacaagctggaa caactgctga agcaagggcg catgtccaag 1200 gaggagatcg attcgacatacaagatcatg aggcgtatcg agggtgagga gctggccctg 1260 gatgcgtcag agcttgtaatcacgagcaca aggcaggaga ttgatgagca gtggggattg 1320 tacgatggat ttgatgtcaagcttgagaaa gtgctgaggg cacgggcgag gcgcggggtt 1380 agctgccatg gtcgttacatgcctaggatg gtggtgattc ctccgggaat ggatttcagc 1440 aatgttgtag ttcatgaagacattgatggg gatggtgacg tcaaagatga tatcgttggt 1500 ttggagggtg cctcacccaagtcaatgccc ccaatttggg ccgaagtgat gcggttcctg 1560 accaaccctc acaagccgatgatcctggcg ttatcaagac cagacccgaa gaagaacatc 1620 actaccctcg tcaaagcgtttggagagtgt cgtccactca gggaacttgc aaaccttact 1680 ctgatcatgg gtaacagagatgacatcgac gacatgtctg ctggcaatgc cagtgtcctc 1740 accacagttc tgaagctgattgacaagtat gatctgtacg gaagcgtggc gttccctaag 1800 catcacaatc aggctgacgtcccggagatc tatcgcctcg cggccaaaat gaagggcgtc 1860 ttcatcaacc ctgctctcgttgagccgttt ggtctcaccc tgatcgaggc tgcggcacac 1920 ggactcccga tagtcgctaccaagaatggt ggtccggtcg acattacaaa tgcattaaac 1980 aacggactgc tcgttgacccacacgaccag aacgccatcg ctgatgcact gctgaagctt 2040 gtggcagaca agaacctgtggcaggaatgc cggagaaacg ggctgcgcaa catccacctc 2100 tactcatggc cggagcactgccgcacttac ctcaccaggg tggccgggtg ccggttaagg 2160 aacccgaggt ggctgaaggacacaccagca gatgccggag ccgatgagga ggagttcctg 2220 gaggattcca tggacgctcaggacctgtca ctccgtctgt ccatcgacgg tgagaagagc 2280 tcgctgaaca ctaacgatccactgtggttc gacccccagg atcaagtgca gaagatcatg 2340 aacaacatca agcagtcgtcagcgcttcct ccgtccatgt cctcagtcgc agccgagggc 2400 acaggcagca ccatgaacaaatacccactc ctgcgccggc gccggcgctt gttcgtcata 2460 gctgtggact gctaccaggacgatggccgt gctagcaaga agatgctgca ggtgatccag 2520 gaagttttca gagcagtccgatcggactcc cagatgttca agatctcagg gttcacgctg 2580 tcgactgcca tgccgttgtccgagacactc cagcttctgc agctcggcaa gatcccagcg 2640 accgacttcg acgccctcatctgtggcagc ggcagcgagg tgtactatcc tggcacggcg 2700 aactgcatgg acgctgaaggaaagctgcgc ccagatcagg actatctgat gcacatcagc 2760 caccgctggt cccatgacggcgcgaggcag accatagcga agctcatggg cgctcaggac 2820 ggttcaggcg acgctgtcgagcaggacgtg gcgtccagta atgcacactg tgtcgcgttc 2880 ctcatcaaag acccccaaaaggtgaaaacg gtcgatgaga tgagggagcg gctgaggatg 2940 cgtggtctcc gctgccacatcatgtactgc aggaactcga caaggcttca ggttgtccct 3000 ctgctagcat caaggtcacaggcactcagg tatctttccg tgcgctgggg cgtatctgtg 3060 gggaacatgt atctgatcaccggggaacat ggcgacaccg atctagagga gatgctatcc 3120 gggctacaca agaccgtgatcgtccgtggc gtcaccgaga agggttcgga agcactggtg 3180 aggagcccag gaagctacaagagggacgat gtcgtcccgt ctgagacccc cttggctgcg 3240 tacacgactg gtgagctgaaggccgacgag atcatgcggg ctctgaagca agtctccaag 3300 acttccagcg gcatgtgaatttgatgcttc ttttacattt tgtccttttc ttcactgcta 3360 tataaaataa gttgtgaacagtaccgcggg tgtgtatata tatattgcag tgacaaataa 3420 aacaggacac tgctaactatactggtgaat atacgactgt caagattgta tgctaagtac 3480 tccatttctc aatgtatcaatcggaattc 3509

What is claimed is:
 1. A substantially purified nucleic acid sequencecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:7, the complement of SEQ ID NO:7, SEQ ID NO:10, and thecomplement of SEQ ID NO:10.
 2. The substantially purified nucleic acidsequence of claim 1, wherein said nucleotide sequence is double-strandedand has promoter activity.
 3. The substantially purified nucleic acidsequence of claim 2, wherein said promoter activity is constitutive. 4.A substantially purified nucleic acid sequence comprising theHindIII/XbaI fragment isolated from plasmid pubi9-GUS contained inEscherichia coli cells deposited as NRRLB-30116.
 5. A transgenic plantcell comprising a nucleic acid sequence comprising a double-strandednucleotide sequence listed as SEQ ID NO:10, wherein said nucleotidesequence is operably linked to a nucleic acid sequence of interest.
 6. Amethod for expressing a nucleic acid sequence of interest in a plantcell, comprising: a) providing: i) a plant cell; ii) a nucleic acidsequence of interest; and iii) a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 10, and the complement of SEQ ID NO:10;b) operably linking said nucleic acid sequence of interest to saidnucleotide sequence to produce a transgene; and c) introducing saidtransgene into said plant cell to produce a transgenic plant cell underconditions such that said nucleic acid sequence of interest is expressedin said transgenic plant cell.
 7. A method for expressing a nucleic acidsequence of interest in a plant cell, comprising: a) providing: i) aplant cell; ii) a nucleic acid sequence of interest; and iii) a promotercomprising SEQ ID NO:10; b) operably linking said nucleic acid sequenceof interest to said promoter to produce a transgene; c) introducing saidtransgene into said plant cell to produce a transgenic plant cell underconditions such that said nucleic acid sequence of interest is expressedin said transgenic plant cell, and d) identifying said transgenic plantcell.
 8. The method of claim 6, further comprising d) regeneratingtransgenic plant tissue from said transgenic plant cell.
 9. The methodof claim 6, further comprising d) regenerating a transgenic plant fromsaid transgenic plant cell.